Substituted guanidines having high binding to the sigma receptor and the use thereof

ABSTRACT

The invention relates to a method for the treatment or prophylaxis of psychosis, depression, hypertension, or anxiety in an animal by administering an effective amount of an N,N&#39;-disubstituted guanidine or 2-iminoimadazolidine having an affinity for the sigma receptor. The invention also relates to the novel guanidines of the invention as well as pharmaceutical compositions thereof.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a continuation-in-part of U.S. applicationSer. No. 07/657,759, filed Feb. 21, 1991, now abandoned, and acontinuation-in-part of U.S. application Ser. No. 07/528,216, nowabandoned, filed May 25, 1990, the disclosures of which are fullyincorporated by reference.

FIELD OF THE INVENTION

The invention is in the field of medicinal chemistry. In particular, theinvention relates to N,N'-disubstituted guanidines andN,N'-disubstituted 2-iminoimidazolidines which have high binding to thesigma receptor, pharmaceutical compositions thereof, and methods fortreating or preventing psychotic mental illness, depression,hypertension, and anxiety in animals.

BACKGROUND OF THE INVENTION

Recently, the inventors have described a series of di-arylguanidineswhich are potent ligands for brain sigma receptors (Weber, et al., PNAS(USA) 83:8784-8788 (1986); Campbell et al., J. Neurosci. 9:3380-3391(1989); U.S. Pat. No. 4,709,094). Brain sigma receptors bind manypsychotropic drugs (Sonders et al., Trends Neurosci. 11:37-40 (1988)).The physiological function of sigma receptors in the nervous system issubject to intense investigations (Sonders et al., Trends Neurosci.11:37-40 (1988)) because certain sigma receptor selective compounds haveknown antipsychotic activity suggesting that sigma receptor activecompounds can be used for the treatment of schizophrenia (Largent etal., Eur. J. Pharmacol., 11:345-347 (1988)).

A wide variety of substituted guanidines are disclosed in the patentliterature. For example:

U.S. Pat. Nos. 1,411,731 and 1,422,506 discloses diphenylguanidine as arubber accelerator;

U.S. Pat. No. 1,597,233 discloses N-o-tolyl-N'-phenyl-guanidine as arubber accelerator;

U.S. Pat. No. 1,672,431 discloses N,N'-di-o-methoxyphenyl-guanidine asbeing useful for therapeutic purposes, especially in the form ofwater-soluble salts;

U.S. Pat. No. 1,730,338 disclosesN-p-dimethyl-amino-phenyl-N'-phenylguanidine as a rubber accelerator;

U.S. Pat. No. 1,795,738 discloses a process for the production ofN,N'-dialkyl-di-substituted guanidines, includingN-di-ethyl-N'-phenyl-guanidine, N-diethyl-N-isoamylguanidine,N-dimethyl-N'-isoamylguanidine and N-dimethyl-N'-ethylguanidine;

U.S. Pat. No. 1,850,682 discloses a process for the preparation ofdisubstituted guanidine rubber accelerators bearing an additionalsubstituent on the imine nitrogen atom;

U.S. Pat. No. 2,145,214 discloses the use of disubstituted guanidines,e.g., diarylguanidines especially dixylylguanidine, as parasiticides;

U.S. Pat. No. 2,254,009 discloses sym-di-2-octyl-guanidine and U.S. Pat.Nos. 2,274,476 and 2,289,542 disclose sym-dicyclohexylguanidine asinsecticides and moth larvae repellents;

U.S. Pat. No. 2,633,474 discloses 1,3-bis(o-ethylphenyl)guanidine and1,3-bis(p-ethylphenyl)guanidine as rubber accelerators;

U.S. Pat. No. 3,117,994 discloses N,N',N"-trisubstituted guanidines andtheir salts as bacteriostatic compounds;

U.S. Pat. No. 3,140,231 discloses N-methyl- andN-ethyl-N'-octylguanidines and their salts as antihypertensive agents;

U.S. Pat. No. 3,248,246 describes (Example 5) a 1,3-disubstitutedguanidine whose substituents are hydrophobic hydrocarbon groups, one ofwhich is naphthylmethyl and the other is n-butyl;

U.S. Pat. No. 3,252,816 discloses various N-substituted andunsubstituted cinnamyl-guanidines and generically the corresponding N'-and N"-alkyl substituted compounds and their salts as antihypertensiveagents;

U.S. Pat. No. 3,270,054 discloses N-2-adamant-1-yl- andN-2-homoadamant-1-yl-oxy-ethyl-thioethyl- and aminoethyl-guanidinederivatives bearing at most two lower alkyl groups on the N'- and/orN"-nitrogen atom as sympathicolytic and anti-viral agents;

U.S. Pat. No. 3,301,755 discloses N-ethylenicallyunsubstituted-alkyl-guanidines and the corresponding N'- and/or N"-loweralkyl compounds as hypoglycemic and antihypertensive agents;

U.S. Pat. No. 3,409,669 disclosesN-cyclohexylamino-(3,3-dialkyl-substituted-propyl)-guanidines and thecorresponding N'-alkyl- and/or N"-alkyl-substituted compounds ashypotensive agents;

U.S. Pat. No. 3,547,951 discloses 1,3-dioxolan-4-yl-alkyl-substitutedguanidines which have anti-hypertensive activity and discloses loweralkyl, including n-butyl, as a possible substituent on the other aminogroup;

U.S. Pat. No. 3,639,477 discloses propoxylguanidine compounds as havinganorectic properties;

U.S. Pat. Nos. 3,681,459; 3,769,427; 3,803,324; 3,908,013; 3,976,787;and 4,014,934 disclose aromatic substituted guanidine derivativeswherein the phenyl ring can contain hydroxy and/or halogen substituentsfor use in vasoconstrictive therapy;

U.S. Pat. No. 3,804,898 discloses N-benzylcyclobutenyl andN-benzylcyclo-butenyl-alkyl-guanidines and the corresponding N-alkyland/or N"-alkyl-substituted compounds as hypotensive agents;

U.S. Pat. No. 3,968,243 discloses N-axalkyl substituted guanidines andthe corresponding N'-alkyl-n"alkyl and N',N'-aralkyl compounds as beinguseful in the treatment of cardiac arrhythmias;

U.S. Pat. No. 3,795,533 discloses o-halo-benzylidene-amino-guanidinesand their use as anti-depressants for overcoming psychic depression;

U.S. Pat. No. 4,007,181 discloses various N,N'-disubstituted guanidinessubstituted on the imine nitrogen atom by adamantyl as possessingantiarrhythmic and diuretic activities;

U.S. Pat. No. 4,051,256 discloses N-phenyl- andN-pyridyl-N'-cycloalkylguanidines as antiviral agents;

U.S. Pat. Nos. 4,052,455 and 4,130,663 disclose styrylamidines, asanalgesics agents or for the prevention of blood platelet aggregation;

U.S. Pat. No. 4,109,014 discloses N-hydroxysubstituted guanidines andthe corresponding N-methyl disubstituted guanidines as vasoconstrictoragents;

U.S. Pat. No. 4,169,154 discloses the use of guanidines in the treatmentof depression;

U.S. Pat. No. 4,393,007 discloses N-substituted and unsubstituted,N-substituted methyl-N'-unsubstituted, monosubstituted anddisubstituted-N"-unsubstituted and substituted guanidines as ganglionicblocking agents; and

U.S. Pat. No. 4,471,137 discloses N,N,N'N"-tetraalkyl guanidines asbeing sterically hindered bases useful in chemical synthesis.

U.S. Pat. No. 4,709,094 discloses 1,3-disubstituted-guanidines, e.g.,1-3-dibutylguanidine and 1,3 di-o-tolyl-quinidine, as sigma brainreceptor ligands.

For examples of other substituted guanidines, see, e.g., U.S. Pat. Nos.1,422,506; 1,642,180; 1,756,315; 3,159,676; 3,228,975; 3,248,426;3,283,003; 3,320,229; 3,479,437; 3,547,951; 3,639,477; 3,784,643;3,949,089; 3,975,533; 4,060,640 and 4,161,541.

Geluk, H. W., et al., J. Med. Chem., 12, 712 (1969) describe thesynthesis of a variety of adamantyl disubstituted guanidines as possibleantiviral agents, including N,N'-di-(adamantan-1-yl)-guanidinehydrochloride, N-(adamantan-1-yl)-N'-cyclohexyl-guanidine hydrochlorideand N-(adamantan-1-yl)-N'-benzyl-guanidine hydrochloride.

U.S. Pat. No. 4,709,094 (1987), discloses N,N'-disubstituted guanidinederivatives which exhibit high binding activity with respect to thesigma receptor having the Formula (I): ##STR1## wherein R and R' are analkyl group of at least 4 carbon atoms, a cycloalkyl group of 3-12carbon atoms, or carbocyclic or aryl, of at least 6 carbon atoms.

Two of the novel N,N'-disubstituted guanidines disclosed therein arealso claimed therein viz., 1,3-di-(4-halo-2-methylphenyl)-guanidine and1,3-di-(4-³ H]-(2-methylphenyl)-guanidine.

Also claimed therein is a method of determining the relationship ofabnormal psychotic-like behavior in a mammal displaying such behavior tosigma receptor system dysfunction, which comprises administering to themammal displaying such behavior a water-solubleN,N'-disubstituted-guanidine which displaces in vitro N,N'-di-(4-[³H]-2-methylphenyl)-guanidine bound to mammalian brain membrane, in anamount effective to alter the sigma brain receptor-modulated mentalactivity of the mammal; a method of treating a human being sufferingfrom a psychotic mental illness associated with hallucinations, whichcomprises administering thereto a water-soluble N,N'-disubstitutedguanidine which is an antagonist to the sigma receptor binding activityof a hallucinogenic benzomorphan, in an amount effective to amelioratethe hallucinations.

In U.S. Pat. No. 4,709,094 is further disclosed a method of determiningthe sigma brain receptor binding activity of an organic compound whichcomprises the steps of a) contacting in an aqueous medium a known mountof isolated mammalian brain membrane which has sigma receptor-likebinding activity, with a mixture of (i) a tritium labeledN,N'-disubstituted guanidine which selectively binds sigma brainreceptors, in a known mount capable of being bound to the sigmareceptors of that brain membrane; and (ii) varying known amounts of awater soluble organic compound to be assayed for sigma receptor bindingactivity; b) separating the brain membrane from the tritium labeledcompound which is not bound to the brain membrane in step a); and c)determining, from the molar relationship of the proportion of boundtritium-labeled compound which is separated in step b) to the molaramount of the organic compound employed in step a), the sigma receptorbinding activity of that organic compound.

Certain benzomorphan opiates, such as N-allyl-normetazocine (SKF 10,047)and cyclazocine, in addition to analgesia, cause hallucinations,depersonalization, drunkenness and other psychotomimetic effects in man.In monkeys, dogs and rodents the psychotomimetic opiates causebehavioral and autonomic effects that are unlike those observed withadministration of classical opiates such as morphine or the opioidpeptides. Specific sigma "opioid" receptors in the brain are believed tomediate such atypical effects. Martin et al., J. Pharmacol. Exp. Ther.197:517-532 (1976). It is believed that the sigma receptors also mediatesome of the psychotomimetic effects of phencyclidine [PCP, angel dust],or alternatively, that psychotomimetic opiates act at specific PCPreceptors. Zukin, R. S., et al., Mol. Pharmacol. 20:246-254 (1981);Shannon, H. E., J. Pharmacol. Exp Ther. 225:144-152 (1983); White, J.M., et al., Psycho-pharmacology 80:1-9 (1983); and Zukin et al., J.Neurochem. 46:1032-1041 (1986). PCP is a drug of abuse that causes abehavioral syndrome in man similar to that which is observed inschizophrenic psychosis. Aniline, O., et al., CRC Critical Rev. Toxicol.10:145-177 (1982). Because of the potent psychotomimetic effects ofsigma opiates and PCP, it is believed that sigma (and/or PCP) receptorsplay a role in mental illness, particularly schizophrenia.

A systematic investigation of the role of sigma receptors in normal andabnormal brain function has been hindered by a lack of specific sigmareceptor binding assays and bioassays. Development of such specificassays requires well-characterized, highly selective and potent sigmareceptor ligands. Recent studies have shown that brain membranereceptors can be labeled in vitro with (±)[³ H]SKF 10,047, Su, T. P., J.Pharmacol. Exp. Ther. 223:284-290 (1982); (+)[³ H]SKF 10,047, Tam, S.W., et al., Proc. Natl. Acad. Sci. U.S.A. 81:5618-5621 (1984); Martin etal., J. Pharmacol. Exp. Ther. 231:539-544 (1984); and Mickelson, M. M.,et al., Res. Commun. Chem. Pathol. Pharmacol. 47:255-263 (1985),although not selectively, Gundlach et al., Eur. J. Pharmacol.113:465-466 (1985); and Largent, B. L., et al., J. Pharmacol. Exp. Ther.238:739-748 (1986), and with (+)[³H]3-(3-hydroxyphenyl)-N-(1-propyl)-piperidine ((+)[³ H]3-PPP), Largentet al., Proc. Natl. Acad. Sci. U.S.A. 81:4983-4987 (1984), which isapparently more selective for sigma receptors than the others.

After the initial in vitro studies by Martin et al., (1976) supra, Keatsand Telford (Keats, A. S., et al., "Analgesics: Clinical Aspects." InMolecular Modification in Drug Design, R. F. Gould (ed.), Advances inChemistry Series #45 Amer. Chem. Soc., Wash. D.C. (1964)), and Haertzen(Haertzen, C. A. Cyclazocine and Nalorphine on the Addiction ResearchCenter Inventory (ARCI), Psychopharmacologia (Berl.) 18:355-377 (1970)),numerous investigators set out to biochemically characterize thedifferent opiate receptors (mu receptors, kappa receptors and sigmareceptors) in vitro.

The first evidence for the existence of a separate sigma receptor intest tube experiments was provided by Su (1982) supra in a paperdescribing an etorphine-inaccessible binding site in guinea pig brainmembranes which was apparently selectively labeled by tritium-labeledSKF-10,047. To overcome the fact that SKF10,047 could label multipleopioid receptors in the brain, Su performed his receptor binding assayusing tritium labeled SKF-10,047 in the presence of excess unlabeledetorphine. Etorphine is a very strong opiate agonist drug which is knownto bind to delta receptors, mu receptors and kappa receptors with almostequal potency. Su used etorphine to saturate all mu, kappa and deltareceptors in a brain membrane preparation and then added tritium labeledSKF-10,047. This enabled him to detect a sigma binding site that wasapparently different from mu, kappa and delta receptors.

A major breakthrough in identifying the sigma receptor as a separateentity occurred when Tam et al. (1984), supra, demonstrated that theprevious problems in selectively labeling the sigma receptor were causedby the fact that in all previous experiments a racemic SKF-10,047preparation was used. Tam showed that using a tritium labeled(+)-SKF-10,047 isomer one could selectively label a sigma receptor thatwas different from the mu, delta and kappa opioid receptors. On theother hand, Tam showed that (-)-SKF-10,047 apparently labeled the mu andkappa receptors but not the sigma receptors. Tam, S. W., Eur. J. Pharm.109:33-41 (1985). This finding has now been confirmed. (Martin et al.,1984, supra). Moreover, there is evidence from behavioral experiments,Khazan et al., Neuropharm. 23:983-987 (1984); Brady et al., Science215:178-180 (1981), that it is the (+)-SKF-10,047 isomer that is solelyresponsible for the psychotomimetic effects of SKF-10,047.

One of the most important findings of the biochemical characterizationof the sigma receptor has been that this receptor binds all syntheticopiate drugs that are known to have hallucinogenic and psychotomimeticeffects. Opiates that do not have psychotomimetic effects in vivo do notbind to this receptor. Most importantly, it has been shown that besideshallucinogenic opiate drugs, the sigma receptor also binds manyantipsychotic drugs that are used clinically to treat hallucinations inschizophrenic patients. (Tam and Cook, 1984). The initial observationswith regard to antipsychotic drug binding to the sigma receptor (Su,1982) were subsequently extensively confirmed and extended by Tam et al.(1984), supra, also showed that when one used radioactively labeledhaloperidol, one of the most potent antipsychotic drugs that is usedclinically, about half of the binding sites in brain membranepreparations are actually sigma receptors whereas the other half of thebinding sites are apparently dopamine receptors. It has long been knownthat most antipsychotic drugs are also dopamine receptor antagonists.Previously the beneficial actions of antipsychotic drugs in psychoticpatients have been attributed to the dopamine receptor-blocking effectof these drugs. It is clear from the work by Tam, however, that numerousclinically used antipsychotic drugs also bind to the sigma site. Allantipsychotic drugs that bind to the sigma receptor may in part causethe beneficial effect of alleviating hallucinations through the sigmareceptor. Taken together all these observations suggest the sigmareceptor as a prime candidate to be involved in the pathogenesis ofmental illness, particularly schizophrenia in which hallucinations are amajor clinical symptom.

Deutsch, S. I., et al. (Clinical Neuropharmacology, Vol. 11, No. 2, pp.105-119 (1988)) provided a review of the literature which implicates thesigma receptor site in psychosis and anti-drug efficacy. According toDeutsch et al., certain benzomorphans which possess analgesic potency inhumans are also associated with a high incidence of psychotomimeticeffects. It has now been concluded that the analgesic action isassociated with the levorotatory isomers of racemic mixtures of thebenzomorphans, while the psychotomimetic effects are attributable to thedextrorotatory isomers in the racemic mixtures. See Haertzen, C. A.,Psychopharmacologia 18:366-77 (1970), and Manallack, D. T., et al.,Pharmacol. Sci. 7:448-51 (1986). Coupled with the fact that many of thein vivo effects of these dextrorotatory enantiomers and the binding ofdextrorotatory tritiated SKF-10,047 are not antagonized by naloxone ornaltrexone, these data strongly support the concept that thepsychotomimetic effects of the dextrorotatory enantiomers are associatedwith the sigma receptor binding site.

Further, Su, T. P., et al. (Life Sci. 38:2199-210 (1986)), andContreras, P. C., et al. (Synapse 1:57-61 (1987)), have established theexistence of endogenous ligands for the sigma receptor, suggesting thatthe dysregulation of the synthesis, release, or degradation of thesenatural ligands may be a naturally occurring mechanism of psychosis.Accordingly, sigma receptor antagonism provides the potential for aneffective antipsychotic therapeutic treatment. See Ferris, R. M., etal., Life Sci. 38:2329-37 (1986), and Su, T. P., Neurosci. Let. 71:224-8(1986).

As further evidence of the role of the sigma receptor in psychosis, thesubstituted carbazolecis-9-[3-(3,5-dimethyl-1-piperazinyl)propyl]carbazole dihydrochloride(rimcazole) was identified as a potential antipsychotic agent based onits ability to antagonize apomorphine-induced mesolimbic behaviorsselectively without altering the intensity of stereotypic behaviors.Further, the compound does not accelerate the rate of dopamine synthesisand does not affect dopamine-stimulated production of cAMP inhomogenates of rat striatum and olfactory tubercle, thus establishingthat rimcazole does not exert its action at the level of post-synapticdopamine receptors in the mesolimbic area.

Rimcazole is able to competitively inhibit the specific binding ofdextrorotatory tritiated SKF-10,047, the prototype sigma receptoragonist, suggesting that rimcazole acts at the sigma receptor site.Rimcazole, therefore, shows potential antipsychotic activity in humans,without extrapyramidal effects, pharmacological behavior which isconsistent with its role as a competitive antagonist of thesigma-receptor.

Another compound, BMY 14802, has demonstrated many properties inpreclinical behavioral tests which suggest its efficacy as a potentialantipsychotic agent which is devoid of extrapyramidal side effects. Thecompound (1) did not cause catalepsy in rats; (2) does not inhibit thebinding of [³ H]spiperone to the D₂ class of striatal dopamine receptorsin rats; (3) did not increase the maximal density of the [³H]spiperone-labeled D₂ site in striatum even following chronicadministration (20 days) to rats; (4) does not appear to interact withthe D₁ subclass of dopamine receptors; and (5) does not inhibitdopamine-stimulated cAMP production or the binding of [³ H]SCH 23390 invitro. These data suggest that BMY 14802 has a low potential forproduction of tardive dyskinesia and further suggests that theantipsychotic effects would be mediated by a nondopaminergic site.Further, BMY 14802 binds with relatively high affinity to the sigmareceptor, with the binding being stereoselective (the dextrorotatoryenantiomer being 10 times more potent at inhibiting binding than thelevorotatory enantiomer). BMY 14802 does not bind to the adrenergic,muscarinic, cholinergic, or histaminergic sites, suggesting that thecompound would not be associated with unpleasant sedative and autonomicside effects.

Accordingly, compounds which bind selectively to the sigma receptor siteand which antagonize this site may be expected to be usefulantipsychotic drugs which are devoid of extrapyramidal effects.

The antipsychotic and anti-schizophrenia drugs that are currently in usehave very strong side effects that are mainly due to their action ondopamine receptors. The side effects often involve irreversible damageto the extrapyramidal nervous system which controls movement functionsof the brain. Patients under long term antischizophrenic drug treatmentoften develop a syndrome that involves permanent damage of their abilityto control coordinated movement.

The foregoing studies have shown that the sigma binding site has thecharacteristics of 1) stereo-selectivity towards dextrorotatorybenzomorphan opiates and insensitivity for naloxone; 2) high affinityfor haloperidol and moderate to high affinity for phenothiazineantipsychotic drugs which are also known to be potent dopamine receptorblockers; and 3) insensitivity for dopamine and apomorphine. Thisintriguing drug selectivity profile calls for a thorough analysis of therole of sigma receptors in normal and abnormal brain function. In orderto do so, it is essential that a spectrum of highly selective and potentsigma receptor active compounds be available. This invention providessuch compounds and methods for identifying other drugs having suchactivity.

Fear, or apprehension, is characterized by the anticipation of a knowndanger or event. In contrast, neurotic anxiety is characterized by anapprehension with no known cause, or a maladaptive response to a trivialdanger. In recent years, generalized anxiety disorder (GAD) has beencharacterized by psychiatrists as being chronic (continually present forat least 1 month) and exemplified by three of four psychomotor symptoms:motor tension, autonomic hyperactivity, apprehensive expectation,vigilance and scanning. Before this characterization was adopted,clinical trials of anxiolytic agents in the U.S. occurred in patientswhich were described variously as suffering from anxiety neurosis,anxiety with associated depression, and other such terms. Anxietydisorders affect 2-3% of the general population (the 67 millionprescriptions written in 1977 for just two popular anxiolytics confirmthis projected incidence). The popularity of anxiolytics attests totheir ability to ameliorate the debilitating symptoms of the disease.Taylor, D. P., FASEB J. 2: 2445-2452 (1988).

Historically, anxiety has been treated by agents including alcoholopiates, and belladonna, which have a sedative component to theiraction. In the 20th century novel chemical entities were discoveredwhich are safer for the treatment of anxiety including barbiturates,propanediol carbamates, and benzodiazepines. The pharmacologicalprofiles of these drugs have suggested that their actions are mediatedby receptors for γ-aminobutyric acid (GABA). Although thebenzodiazepines present a safer alternative than meprobamate andphenobarbital, they also are sedatives. In addition, the benzodiazepinescontrol convulsions and produce muscle relaxation, properties that areunneeded or undesirable in the treatment of anxiety. Furthermore, thesedrugs can interact with alcohol, with potentially disastrousconsequences. Recently, it has been appreciated that the benzodiazepinesproduce habituation and possess a pronounced liability, for example,withdrawal symptoms after chronic use. The need for anxioselective drugsthat are more selective, have fewer side effects, and present a profileconsistent with safety during protracted treatment has resulted in acontinuing search for such drugs. This search has led to the synthesisand evaluation of agents that possess no obvious homology with thebenzodiazepines. Taylor et al., supra.

Buspirone (Buspar) was the first novel anxiolytic to be approved forclinical use in the U.S. since the benzodiazepines were introducedalmost 30 years ago. The introduction of buspirone into clinical trialsfor the treatment of anxiety was a direct result of its efficacy in apredictive animal model for the disease--the taming of the aggressiveresponse of rhesus monkeys to the introduction of foreign objects intotheir cages according to the protocol described by Tompkins, E. C. etal., Res Commun. Psychol. Psychiatry Behav. 5: 337-352 (1980). SeeTaylor et al., supra.

The preclinical screening of putative anxiolytics is dependent uponanimal tests. Most of the laboratory data on new putative anxiolyticscome from animal tests form two main classes. The first group of testsare based on conflict or conditioned fear. The second group of tests arebased upon anxiety generated by novel situations. Although these testsdiffer in the way anxiety is produced, there has been surprisingagreement amongst them in the classification of drugs as anxiolytic oranxiogenic. See File, S. E., TINS 10:461-463 (1987).

In two particular tests for anxiolytic activity, it is assumed that theanticipation of punishment causes a reduction in a response associatedwith the punishment. Conversely, anxiolytic agents that reduce anxietyresult in an increased response rate. In the Geller-Seifter test, therat receives food reward for pressing a lever, but also receives anelectric footshock, which has the effect of suppressing the response.This punished schedule alternates with an unpunished schedule whereinelectric footshocks are not administered. During this unpunishedschedule, lever-pressing is still rewarded. In the Vogel test, a rat isallowed to drink water, but also receives an electric shock through thewater spout or the bars of the floor. In both the Vogel andGeller-Seifter tests, a measure of unpunished response is obtained inorder to allow assessment of any non-specific stimulant or sedative drugeffects or any changes in food or water intake. In both of these tests,benzodiazepines enhance the response rate in the punished periods,without increasing the rate of response in the absence of shock. Whilethese tests are valid tests of anxiety, the only means of assessing themhas been pharmacological. Taylor et al., supra.

A less widely used test utilizes punished locomotion, wherein a measureof unpunished crossing is obtained according to the rate at which amouse crosses from one metal plate to another, wherein footshocks areadministered whenever the mouse crosses. Although less widely utilizedto test anxiolytic agents, this test has been able to detectdrug-induced increases and decreases in anxiety by manipulating theshock level. File, S. E., J. Neurosci Methods 2:219-238 (1980).

The social interaction test of anxiety (File, supra; Jones, B. J. etal., Br. J. Pharmacol. 93:985-993 (1988) exploits the uncertainty andanxiety generated by placing rats in an unfamiliar environment and inbright light. The dependent variable is the time that pairs of male ratsspend in active social interaction (90% of the behaviors areinvestigatory in nature). Both the familiarity and the light level ofthe test arena may be manipulated. Undrugged rats show the highest levelof social interaction when the test arena is familiar and is lit by lowlight. Social interaction declines if the arena is unfamiliar to therats or is lit by bright light. Anxiolytic agents prevent this decline.The overall level of motor activity may also be measured to allowdetection of drug effects specific to social behaviors.

The social interaction test of anxiety is one of the few animal tests ofanxiety that has been validated behaviorally. Other behavioral measuresindicative of anxiety and stress (e.g. defecation, self-grooming anddisplacement activities) were correlated with the reductions in socialinteraction; and other causes of response change (e.g. exploration ofthe environment, odor changes) were excluded. In order to validate thetest physiologically, ACTH and corticosterone levels and changes inhypothalamic noradrenaline also were measured. File, TINS 10: 461-463(1987).

Another test of anxiety that exploits the anxiety generated by a novelsituation is the "elevated plus maze." In this test, the anxiety isgenerated by placing the animals on an elevated open arm. Height, ratherthan the light level, is responsible for generating behavioral andphysiological changes. The apparatus is in the shape of a plus with twoopen and two enclosed arms. The rat has free access to all arms on theapparatus. Anxiolytic activity may be measured by the percentageincrease in the time that the test animal spends on the open arms andthe number of entries onto the open arms. This test has also beenvalidated behaviorally and physiologically.

Agents which have been determined to have anxiolytic activity includethe carbazole derivative9-[3-(3,5-cis-dimethylpiperazino)propyl]carbazole having the Formula(II): ##STR2## and pharmaceutical compositions thereof. See U.S. Pat.No. 4,400,383 (1983).

Serotonin receptor antagonists are also known to be useful for thetreatment of anxiety. See Kahn, R. S. et al., J. Affective Disord.8:197-200 (1987); Westenberg, H. G. M. et al., Psychopharmacol. Bull.23:146-149 (1987).

SUMMARY OF THE INVENTION

The invention relates to novel N,N'-disubstituted guanidines which bindto sigma receptor sites, especially those which do so selectively.

The invention also relates to a novel class of N,N'-disubstitutedguanidines which are radioactively tagged and which are useful forassaying in vitro the sigma receptor binding activity of organiccompounds.

The invention also relates to pharmacological compositions comprisingcertain of the aforesaid N,N'-disubstituted guanidines having sigmareceptor binding activity and the use thereof to treat or preventpsycosis, depression, hypertension or anxiety.

The invention also relates to a method for determining the sigmareceptor binding activity of organic compounds.

The invention also relates to an in vitro screening method for assayingcompounds having sigma receptor activity and utility as antipsychotic,antidepressant, antihypertensive and anxiolytic drugs.

The invention also relates to a method of determining the relationshipof abnormal psychotic-like behavior in a mammal displaying such behaviorto sigma receptor system dysfunction.

The substituted guanidines of the invention have the Formula (I):##STR3## wherein R and R' are an alkyl group of at least 4 carbon atoms,a cycloalkyl group of at least 3 carbon atoms, a carbocyclic aryl groupof at least 6 carbon atoms, alkaryl or aralkyl of at least 6 carbonatoms and containing 1-3 separate or fused rings, a heterocyclic ring ora heteroaryl group, and wherein each of R and R' may be substituted in1-3 positions, or wherein R and R' together with the guanidine group towhich they are attached form a saturated or unsaturated cyclic ringcontaining at least 2 carbon atoms exclusive of the guanidine carbonatom, and wherein said cyclic ring may be substituted with one or morealkyl groups of 1-6 carbon atoms, carbocyclic aryl groups of at least 6carbon atoms, cycloalkyl groups of 3-12 carbon atoms, or 1-2 fusedaromatic rings, and further wherein said N,N'-disubstituted guanidineexhibits a high affinity for the sigma receptor. Preferably, suchN,N'-disubstituted guanidines also exhibit activity in one or more ofthe animal models disclosed herein.

In particular, the invention relates to N,N'-disubstituted guanidines ofthe Formula (I), above, wherein R and R' each are adamantyl, cyclohexyl,coumarinyl, norbornyl, isobornyl, or a monocyclic carbocyclic aryl of atleast 6 carbon atoms.

The invention also relates to substituted guanidines having the Formula(III): ##STR4## wherein X and Y are independently a branched or straightchain C₁ -C₁₂ alkylene or a branched or straight chain C₂ -C₁₂unsaturated alkylene or wherein one of X and Y is a single bond;

R and R' are independently hydrogen, a cycloalkyl group of at least 3carbon atoms, a carbocyclic aryl group of at least 6 carbon atoms,aralkyl of at least 6 carbon atoms and containing 1-3 separate or fusedrings, a heterocyclic ring or a heteroaryl group, and wherein each of Rand R' may be substituted in 1-3 positions, or wherein R and R' togetherwith the guanidine group to which they are attached form a saturated orunsaturated cyclic ring containing at least 2 carbon atoms exclusive ofthe guanidine carbon atom, and wherein said cyclic ring may besubstituted with one or more alkyl groups of 1-6 carbon atoms,carbocyclic aryl groups of at least 6 carbon atoms, cycloalkyl groups of3-12 carbon atoms, or 1-2 fused aromatic rings. Preferably, saidN,N'-disubstituted guanidine exhibits a high affinity for the sigmareceptor.

Preferably, X and Y are C₁ -C₄ alkylene groups. Also contemplated foruse in the claimed invention are the N,N'-disubstituted guanidineshaving Formulae (I) and (III), wherein one or two C₁ -C₆ lower alkylgroups are substituted on N and/or N'.

The invention also relates to compounds having the Formula (IV):##STR5## wherein n is 2, 3, 4 or 5;

X and Y are independently a single bond, a branched or straight chain C₁-C₁₂ alkylene or a branched or straight chain C₂ -C₁₂ alkylene;

R and R' are independently hydrogen, a cycloalkyl group of at least 3carbon atoms, a carbocyclic aryl group of at least 6 carbon atoms,aralkyl of at least 6 carbon atoms and containing 1-3 separate or fusedrings, a heterocyclic ring, and wherein each of R and R' may besubstituted in 1-3 positions, or wherein R and R' together with theguanidine group to which they are attached form a saturated orunsaturated cyclic ring containing at least 2 carbon atoms exclusive ofthe guanidine carbon atom, and wherein said cyclic ring may besubstituted with one or more alkyl groups of 1-5 carbon atoms,carbocyclic aryl groups of at least 6 carbon atoms, cycloalkyl groups of3-12 carbon atoms, or 1-2 fused aromatic rings. Preferably, saidcompound exhibits a high affinity for the sigma receptor. Alsopreferable is where R and R' are m-ethylphenyl, X and Y are single bondsand n is 2, e.g., N,N'-di(m-ethylphenyl)-2-iminoimidazolidine.

The invention also relates to tritiated derivatives of the above-listedN,N'-disubstituted guanidines wherein at least one of the ring carbonatoms of R and R' bears at least one radioactive atom, preferably atritium atom

The invention also relates to a method of determining the sigma brainreceptor binding activity of an organic compound which comprises thesteps of:

a) contacting in an aqueous medium a known amount of isolated mammalianbrain membrane which has sigma receptor-type binding activity, with amixture of (i) a radio-labeled N,N'-disubstituted guanidine whichselectively binds sigma brain receptors, in a known amount capable ofbeing bound to the sigma receptors of that brain membrane; and (ii)varying known amounts of a water soluble organic compound to be assayedfor sigma receptor binding activity;

b) separating the brain membrane from the radio-labeled compound whichis not bound to the brain membrane in step a);

c) determining, from the molar relationship of the proportion of boundradio-labeled compound which is separated in step b) to the molar amountof the organic compound employed in step a), the sigma receptor bindingactivity of that organic compound.

The invention also relates to a method of determining the relationshipof abnormal psychotic-like behavior in a mammal displaying such behaviorto sigma receptor dysfunction, which comprises administering thereto asigma brain receptor-modulating amount of a water-solubleN,N'-disubstituted guanidine which displaces in vitro N,N'-di-(4-[³H]-2-methylphenyl)-guanidine bound to mammalian brain membrane,effective to alter the sigma brain receptor-modulated mental activity ofthat mammal.

By the present invention, there is provided a means for identifyingcompounds which bind competitively and selectively to the sigma receptorsite. Accordingly, the invention provides compounds, pharmaceuticalcompositions, and the use of same for treatment of psychoses,depression, hypertension or anxiety. Selective sigma receptor binding isof particular value in reducing or eliminating undesirableextrapyramidal side effects associated with present antipsychoticmedications.

It has been discovered further that certain N,N'-disubstitutedguanidines are potent anxiolytics, and at the same time, aresubstantially non-sedative in an animal model. Therefore, theseN,N'-disubstituted guanidines are useful for the treatment orprophylaxis of anxiety in animals, i.e., humans.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graphical representation of the data resulting from the invivo subcutaneous (s.c.) administration of diazepam on mouse behavior inthe black:white test box.

FIG. 2 is a graphical representation of the data resulting from the invivo s.c. administration of N,N'-di(adamantan-1-yl)guanidine (DAG) onmouse behavior in the black:white test box.

FIG. 3 is a graphical representation of the data resulting from the invivo s.c. administration ofN-(2-methylphenyl)-N'-(adamantan-1-yl)guanidine (AdTG) on mouse behaviorin the black:white test box.

FIG. 4 is a graphical representation of the data resulting from the invivo oral administration of AdTG on mouse behavior in the black:whitetest box.

FIG. 5 is a graphical representation of the data resulting from the invivo s.c. administration ofN-(2-iodophenyl)-N'-(adamantan-1-yl)guanidine (AdIpG) on mouse behaviorin the black:white test box.

FIG. 6 is a graphical representation of the data resulting from the invivo s.c. administration ofN-(3,5-dimethyladamantan-1-yl)-N'-[(E)-2-phenylethenyl]phenylguanidine(F-114-B) on mouse behavior in the black:white test box.

FIG. 7 is a graphical representation of the data resulting from the invivo s.c. administration of DTG on mouse behavior in the black:whitetest box.

FIG. 8 is a graphical representation of the data resulting from the oraladministration of N,N'-di-(adamantan-1-yl)guanidine andN-(adamantan-1-yl)-N'-(o-iodophenyl)guanidine, in comparison withcontrols, on mouse behavior in the black:white test box.

FIG. 9 is a graphical representation of the data resulting from the invivo s.c. administration of N-cyclohexyl-N'-(o-totyl)guanidine, incomparison to controls, on mouse behavior in the black:white test box.

FIG. 10 is a graphical representation of the data resulting from the invivo s.c. administration ofN-(±)endo-2-norbornyl)-N'-(2-iodophenyl)-guanidine, in comparison tocontrols, on mouse behavior in the black:white test box.

FIG. 11 is a graphical representation of data resulting from the in vivos.c. administration of N-(exo-2-norbornyl)-N'-(2-methylphenyl)guanidine,in comparison to controls, on mouse behavior in the black:white testbox.

FIG. 12 is a graphical representation of data resulting from the in vivos.c. administration of N-(adamantan-1-yl)-N'-cyclohexylguanidine, incomparison to controls, on mouse behavior in the black:white test box.

FIG. 13 is a graphical representation of the data resulting from theoral administration of N-cyclohexyl-N'-(2-methylphenyl)guanidine, incomparison with controls, on mouse behavior in the black:white test box.

FIG. 14 is a graphical representation of the data resulting from theoral administration ofN-((±)-endo-2-norbornyl-N'-(2-iodophenyl)guanidine, in comparison withcontrols, on mouse behavior in the black:white test box.

FIG. 15 is a graphical representation of the data resulting from theoral administration of N-(exo-2-norbornyl)-N'-(2-methylphenyl)guanidine,in comparison with controls, on mouse behavior in the black:white testbox.

FIG. 16 is a graphical representation of the data resulting from the invivo i.p. administration ofN-(2-styrylphenyl)-N'-(2-iodophenyl)guanidine, in comparison withcontrols, on mouse behavior in the black:white test box.

FIG. 17 is a graphical representation of the data resulting from the invivo i.p. administration of diazepam and AdTG (for comparison) in therat social interaction test.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

We have discovered that the disubstituted guanidines of this inventionhave-sigma receptor binding activity, as evidenced by their ability todisplace from guinea pig membrane binding sites [³H]-1,3-di-ortho-tolyl-guanidine(N,N'-di-(4-[³H]-2-methylphenyl)-guanidine ([³ H]--DTG]) which has the Formula (IV):##STR6## This compound binds reversibly, saturably, selectively and withhigh affinity to sigma receptor binding sites in guinea pig brainmembrane homogenates and slide-mounted rat and guinea pig brainsections. We have established that (+)-[³ H]3-PPP binds to the samesites. Availability of the selective sigma ligands of this inventionfacilitates characterization of sigma receptors in vivo and in vitro.

The preferred N,N'-disubstituted guanidines of this invention are thoseof the Formula (I): ##STR7## wherein R and R' each are an alkyl group ofat least 4 carbon atoms or carbocyclic aryl groups of at least 6 carbonatoms, e.g., R and R', which can be the same or different, are alkyl of4 or more carbon atoms, e.g., a 4 to 12, preferably a straight chainalkyl and more preferably a 4 to 8 carbon atom alkyl group, for example,butyl, isobutyl, tert-butyl, amyl, hexyl, octyl, nonyl and decyl;cycloalkyl of 3 to 12 carbon atoms, e.g., cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl, cycloheptyl, 1,4-methylene-cyclohexanyl,adamantyl, norbornyl, isobornyl, cyclopentylmethyl, cyclohexylmethyl, 1-or 2-cyclohexylethyl and 1-, 2- or 3-cyclohexylpropyl; carbocyclic aryl,alkaryl or aralkyl, e.g., of up to 18 carbon atoms and containing 1-3separate or fused aromatic rings, e.g., phenyl, benzyl, 1- and2-phenylethyl, 1-, 2-, or 3-phenylpropyl; o-, m- or p-tolyl,m,m'-dimethylphenyl, o-, m- or p-ethylphenyl, m,m'diethylphenyl,m-methyl-m'-ethylphenyl and o-propylphenyl, naphthyl, 2-naphthyl,biphenyl; and heterocyclic rings, e.g. 3-, 4-, 5-, 6-, 7-, and8-coumarinyl; 2- and 4-pyridyl, pyrrolyl especially 2- and3-N-methylpyrrolyl, pyrrolidinyl, 2- and 3-furanyl, 2- and 3-thiophenyl,2- and 3-benzofuranyl, 2-benzoxazolyl, pyrazinyl, piperazinyl,pyrimidyl, 2-, 4- and 5- thiazolyl, 2-, 4- and 5- oxazolyl, 2-, 4- and5-imidazolyl, 2- and 3-indolyl, and 2-4-benzothiazolyl, thienyl,benzofuranyl and morpholino.

Additionally, 1, 2, 3 or more substituents which do not adversely affectthe activity of the N,N'-disubstituted guanidine moiety may be presenton one or both of the R and R' hydrocarbon groups thereof, e.g., alkylof 1-8 carbon atoms, e.g., methyl, ethyl; hydroxyalkyl of 1-8 carbonatoms; halo, e.g., chloro, bromo, iodo, fluoro; hydroxy; nitro; azido,cyano; isocyanato; amino; lower-alkylamino; di-lower-alkylamino;trifluoromethyl; alkoxy of 1-8 carbon atoms, e.g., methoxy, ethoxy andpropoxy; acyloxy, e.g., alkanoyloxy of 1-8 carbon atoms, e.g., acetoxyand benzoxy; amido, e.g., acetamido, N-ethylcetamido; carbamido, e.g.,carbamyl, N-methylcarbamyl, N,N'-dimethylcarbamyl; etc.

Especially preferred are compounds of Formula I wherein R and R' eachare cyclohexyl, norbornyl, adamantan-1-yl, adamantan-2-yl, 8-coumarinyl,or phenyl groups. Such phenyl groups, which need not necessarily beidentical, may be substituted with one or more of the foregoingsubstituents, for example, in the o-, m- or p- position or the 2,2-,2,4- or 3,5-position, when the phenyl group is disubstituted or R is asherein defined and R' is adamantyl. Specific examples are those whereinR and R' both are phenyl or o-tolyl; R is o-tolyl and R' isp-bromo-o-tolyl, p-CF₃ -o-tolyl, p-iodo-o-tolyl, o-iodo-phenyl,p-azido-o-tolyl, cyclohexyl or adamantyl; and R is phenyl and R' isp-bromo-o-tolyl, p-iodo-o-tolyl, m-nitro-phenyl or o-iodo-phenyl.

The highly active disubstituted guanidines of this invention havesubstantially the same stereoconfiguration as (+)3-PPP with phenyl axialand with SKF 10,047 with the piperidine ring in a skew-boat form andN-allyl axial. This similarity in spacial configuration, i.e., asdemonstrated by computer modeling, of compounds having sigma receptorbinding activity provides a screening technique for predicting theprobable level of sigma receptor binding activity of otherN,N'-disubstituted guanidines. Computer assisted molecular modifying canbe used to determine the molecular similarity of the various species inthree dimensions.

Examples of those compounds which have been isolated and/or prepared andfound to possess the aforesaid in vitro [³ H]DTG displacement activityare N,N'-dibutylguanidine, N,N'-diphenylguanidine,N,N'-di-o-tolylguanidine, N,N'-di-(2-methyl-4-bromo-phenyl)guanidine,N,N'-di-(2-methyl-4-iodophenyl)guanidine,N-(2-methyl-azidophenyl)-N'-(2-methylphenyl)guanidine,N,N'-diadamantylguanidine, N-adamantyl-N'-(2-methylphenyl)guanidine,N-(2-iodophenyl)-N'-(2-methylphenyl)guanidine,N-(2-methyl-4-nitrophenyl)-N'-(2-methylphenyl)guanidine,N,N'-di-(2,6-dimethylphenyl)guanidine,N-(2,6-dimethylphenyl)-N'-N'-(2-methylphenyl)guanidine,N-(adamantyl)-N'-(cyclohexyl)guanidine, N,N'-di(cyclohexyl)guanidine,N-(2-iodophenyl)-N'-(adamantyl)guanidine,N-(2-methylphenyl)-N'-cyclohexyl-guanidine,N-adamantyl-N'-phenylguanidine, N,N'-di-(m-n-propylphenyl)guanidine,N,N'-di-(1-tetralinyl)guanidine,N-(3,5-dimethyl-1-adamantanyl)-N'-(o-tolyl)guanidine,N-(3,5-dimethyl-1-adamantanyl)-N'-(o-iodophenyl)guanidine,N-(1-adamantyl)-N'-(o-nitrophenyl)guanidine,N,N'-di-((±)-endo-2-norbornyl)guanidine,N-(exo-2-isobornyl)-N'-(o-iodophenyl)guanidine,N,N'-di-(exo-2-norbornyl)guanidine,N-(exo-2-isobornyl)-N'-(o-tolyl)guanidine,N-(o-iodophenyl)-N'-(t-butyl)guanidine, N,N'-dibenzylguanidine,N-(adamant-1-yl)-N'-(o-isopropylphenyl)guanidine,N-(adamant-1-yl)-N'-(p-bromo-o-tolyl)guanidine,N-(cyclohexyl)-N'-(p-bromo-o-tolyl)guanidine, andN-(adamant-2-yl)-N'-(p-iodophenyl)guanidine.

Among the compounds tested to date, some of those having the highestsigma receptor binding activity are those of Formula I wherein one of Rand R' is adamantyl and the other is also adamantyl or o-substitutedphenyl. Therefore, the preferred compounds of this invention includethose wherein R and R¹ have those values, i.e., wherein the other of Rand R¹ is, e.g., o-lower alkyl phenyl, wherein alkyl is of 1-4 carbonatoms, e.g., CH₃, C₂ H₅, i-C₃ H₇, o-halophenyl wherein halo is Cl, Br, Ior F, o-nitro-o-amino, o-carbo-lower alkoxy, e.g., COOCH₃, o-amino,e.g., --CONH₂, o-sulfato, o-carboxy, o-acyl, e.g., acetyl, o-CF₃,o-sulfamido and o-lower-alkoxy, e.g., o-methoxy, phenyl or anotherphenyl group ortho substituted by any other substituent of a molecularweight less than 150.

Especially preferred N,N'-disubstituted guanidine compounds which havehigh binding to the sigma receptor includeN-(2-iodophenyl)-N'-(adamant-1-yl)guanidine (AdIpG, IC₅₀ =6.2 nM);N-(o-tolyl)-N'-(adamant-1-yl)guanidine (AdTG, IC₅₀ =7.6 nM);N,N'-di-(adamant-1-yl)guanidine (DAG, IC₅₀ =16.5 nM);N-cyclohexyl-N'-(2-methylphenyl)-guanidine (IC₅₀ =13 nM);N-(adamant-1-yl)-N'-cyclohexylguanidine (IC₅₀ =12.5 nM);N-(adamant-2-yl)-N'-(2-iodophenyl)guanidine (IC₅₀ =3.5 nM);N-(adamant-2-yl)-N'-(2-methylphenyl)guanidine (IC₅₀ =7.0 nM);N-(exo-2-norbornyl)-N'-(2-methylphenyl)guanidine (IC₅₀ =7.0nM);N-((±)-endo-2-norbornyl)-N'-(2-methylphenyl)guanidine (IC₅₀ =6.0 nM);N-(exo-2-norbornyl)-N'-(2-iodophenyl)guanidine (IC₅₀ =4.0 nM);N-((±)-endo-2-norbornyl)-N'-(2-iodophenyl)guanidine (IC₅₀ =5.0 nM);N,N'-di-(o-tolyl)guanidine (IC₅₀ =32 nM);N-(o-tolyl)-N'-(o-iodophenyl)guanidine (IC₅₀ =21 nM);N,N'-di-(p-bromo-o-methylphenyl)guanidine (IC₅₀ =37 nM);N,N'-di-(m-n-propylphenyl)guanidine (IC₅₀ =36 nM);N-(o-tolyl)-N'-(p-nitro-o-tolyl)guanidine (IC₅₀ =37 nM);N,N'-di-(1-tetralinyl)guanidine (IC₅₀ =58 nM);N-(o-tolyl)-N'-(o-xylyl)guanidine (IC₅₀ =70 nM);N,N'-di-(o-xylyl)guanidine (IC₅₀ =90 nM); N,N'-di-(cyclohexyl)guanidine(IC₅₀ =71 nM); N-(3,5-dimethyladamantan-1-yl)-N'-(o-tolyl)guanidine(IC₅₀ =15 nM); N-(3,5-dimethyl-1-adamantanyl)-N'-(o-iodophenyl)guanidine(IC₅₀ =16 nM); N-(1-adamantyl)-N'-(o-nitrophenyl)guanidine (IC₅₀ =30nM); N,N'-di-((±)-endo-2-norbornyl)guanidine (IC₅₀ =16 nM);N-(exo-2-isobornyl)-N'-(o-iodophenyl)guanidine (IC₅₀ =18 nM);N,N'-di-(exo-2-norbornyl)guanidine (IC₅₀ =22 nM);N-(exo-2-isobornyl)-N'-(o-tolyl)guanidine (IC₅₀ =25 nM);N-(o-iodophenyl)-N'-(t-butyl)guanidine (IC₅₀ =20 nM);N,N'-dibenzylguanidine (IC₅₀ =90 nM);N-(adamant-1-yl)-N'-(o-isopropylphenyl)guanidine (IC₅₀ =24 nM);N-(adamant-1-yl)-N'-(p-bromo-o-tolyl)guanidine (IC₅₀ =2.7 nM);N-(cyclohexyl)-N'-(p-bromo-o-tolyl)guanidine (IC₅₀ =5.5 nM);N-(4-azido-2-methylphenyl)-N'-(2-methylphenyl)guanidine (IC₅₀ =20 nM);N-(2-methyl-4-nitro-5-bromophenyl)-N'-(2-methyl-4,5-dibromophenyl)guanidine;N,N'-di-(o-iodophenyl)guanidine (IC₅₀ =13 nM);N,N'-di-(3-methylphenyl)guanidine (IC₅₀ =43 nM);N,N'-di-(m-ethylphenyl)-2-imino-imidazolidine (IC₅₀ =70 nM);N-(4-nitro-2-methylphenyl)-N'-(2-methylphenyl)guanidine (IC₅₀ =37 nM);N-(1-naphthyl)-N'-(2-iodophenyl)guanidine (IC₅₀ =40.1 nM);N,N'-di-(4-indanyl)guanidine (IC₅₀ =28.5 nM);N-(adamantan-1-yl)-N'-(2-trifluoromethylphenyl)guanidine (IC₅₀ =7.44nM); N-(adamantan-1-yl)-N'-(2-methylphenyl)-N'-methylguanidine (IC₅₀=22.6 nM); N-(adamantan-1-yl)-N'-(6-coumarinyl)guanidine (IC₅₀ =21.9nM); N-(adamantan-1-yl)-N'-(2,4-difluorophenyl)guanidine (IC₅₀ =8.92nM); andN-(adamantan-1-yl)-N'-(2-trifluoromethyl-4-fluorophenyl)guanidine (IC₅₀=4 nM).

The level of sigma receptor activity of the disubstituted guanidines canalso be determined in vivo in a discriminative stimulus property testemploying rats trained to discriminate between intraperitonealinjections of cyclazocine (2.0 mg/kg) and saline in a discrete-trialavoidance paradigm with sessions of 20 trials each. For example,ditolylguanidine (DTG) and diphenylguanidine (DPG) were fullysubstitutable for cyclazocine at the same concentrations. (Holtzman, S.G., Emery University, Atlanta, Ga., private communication.)

Although the discussion hereinafter of the experiments below relates tocertain of these selective sigma receptor ligands, viz., theN,N'-disubstituted guanidines of Table I below, the activity and utilityof that compound apply comparably to the other disubstituted guanidineswhich compete with and displace in vitro N,N'-di-(4-[³H]-2-methylphenyl)-guanidine bound in vitro to isolated guinea pig brainmembrane.

In carrying out the sigma receptor binding activity measurement methodof this invention, a known amount of a mammalian brain membrane, e.g.,human or other primate, porcine, rodent, e.g., rat or guinea pig, whichhas SKF 10,047 and like psychotomimetic benzomorphan binding activity iscontacted in a suitable aqueous vehicle, e.g., physiological salinesolution, with a mixture, usually in a solution in a suitable aqueousvehicle of (i) a tritium-labeled N,N'-disubstituted guanidine of thisinvention having sigma receptor binding activity, in an mount capable ofbeing fully bound to the above said amount of membrane and (ii) a watersoluble organic compound whose sigma receptor activity is to be assayed,in known mounts, sufficiently varied to obtain a dose-response curve.The techniques for obtaining a dose-response curve are standard and wellknown to those skilled in the art. Typically, one could employ molaramounts varying as much as from 10⁻⁴ to 10⁴ of the molar amount of thetritium labeled compound present in the mixture, e.g., employing from 10to 120 and preferably 30 to 90 such mixtures.

If the organic compound being assayed has sigma receptor bindingactivity, a portion of the tritium labeled compound which, in theabsence of the organic compound would bind to the membrane remainsunbound and is thus separable from the membrane. The amount whichremains unbound is proportional to the sigma receptor binding activityof the organic compound and the molar ratio thereof in the mixture tothe tritium labeled compound.

The two compounds can be employed at any convenient collectiveconcentration, e.g., from 10⁻⁸ to 10³ nM.

In the next step, the membrane is separated from and washed until freeof the solution in which step (a) is conducted. In the next step, theamount of tritium labeled compound which is thus separated from themembrane is determined, e.g., by measuring the collective radioactivitylevel of the separated solution and wash water and comparing thatradioactivity to that obtained when the foregoing steps are conductedwith the same amount of tritium-labeled N,N'-disubstituted guanidine inthe absence of the organic compound.

In the next step of the method, the activity of sigma receptor bindingactivity of the organic compound is determined from the dose responsecurve thus obtained.

Although tritium radiolabels are preferred, any radiolabel which can besubstituted on the N,N'-disubstituted guanidines of the invention may beemployed, e.g., ¹¹ C, ¹⁴ C, ¹⁸ F, ¹²⁵ I, ¹³¹ I, ¹⁵ N, ³⁵ S, and ³² P.

All of the foregoing steps are conventional and have been employed inthe prior art with other types of ³ H-labeled compounds having sigmareceptor binding activity. The method of this invention is, however,unique in that the tritium-labeled N,N'-disubstituted guanidines of thisinvention are highly selective to binding by the sigma receptors andtherefore will not compete with organic compounds which bind to otherbrain receptors.

In carrying out the method of treatment aspect of this invention, e.g.,treating a human being suffering from a psychotic mental illnessassociated with hallucinations, or suffering from depression,hypertension or anxiety, there is administered thereto an effectiveamount of a water-soluble N,N'-disubstituted guanidine which has highbinding to the sigma receptor. When treating psychosis, the compound isan antagonist to the sigma receptor binding activity of a hallucinogenicbenzomorphan. Preferably, the guanidine is a compound of Formulae I, IIIor IV wherein R and R' each is an alkyl group of at least 4 carbonatoms, a cycloalkyl group of 3 to 12 carbon atoms or a carbocyclic arylgroup of at least 6 carbon atoms. In preferred aspect, the human beingis schizophrenic; in another preferred aspect, the compound isN,N'-di-(2-methylphenyl)-guanidine,N-(adamantyl)-N'-(cyclohexyl)guanidine,N-adamantyl-N'-(2-methylphenyl)guanidine,N-(1-adamantyl)-N'-(o-iodophenyl)guanidine,N-(2-adamantyl)-N'-(o-iodophenyl)guanidine,N-cyclohexyl-N'-(2-methylphenyl)guanidine,N,N'-di-(cyclohexyl)guanidine, N,N'-di-(2-adamantyl)guanidine,N,N'-di-(m-n-propylphenyl)guanidine,N-(o-tolyl)-N'-(p-nitro-o-tolyl)guanidine,N,N'-di-(1-tetralinyl)guanidine, N-(o-tolyl)-N'-(o-xylyl)guanidine,N,N'-di-(o-xylyl)guanidine,N-(3,5-dimethyladmantan-1yl)-N'-(o-tolyl)guanidine,N-(3,5-dimethyladamantan-1-yl)-N'-(o-iodophenyl)guanidine,N-(1-adamantyl)-N'-(o-nitrophenyl)guanidine, (+) and(-)N-(exo-2-norbornyl)-N'-(2-iodophenyl)guanidine, (+) and(-)N-(endonorbornyl)-N'-(o-tolyl)guanidine, (+) and(-)N-(exonorbornyl)-N'-(o-tolyl)guanidine, (+) and(-)N,N'-di(endonorbornyl)guanidine, (+) and(-)N-(exoisobornyl)-N'-(o-iodophenyl)guanidine, (+) and(-)N,N'-di-(exonorbornyl)guanidine,N-(o-iodophenyl)-N'-(t-butyl)guanidine, N,N'-dibenzylguanidine,N-(adamant-1-yl)-N'-(o-isopropylphenyl)guanidine,N-(adamant-1-yl)-N'-(p-bromo-o-tolyl)guanidine,N-cyclohexyl)-N'-(p-bromo-o-tolyl)guanidine,N-(adamant-2-yl)-N'-(p-iodophenyl)guanidine,N,N'-di(o-methylbenzyl)guanidine, N,N'-di(1-adamantanemethyl)guanidine,N-(adamantan-1-yl)-N'-(2-trifluoromethylphenyl)-guanidine,N-(adamantan-1-yl)-N'-(2,4-difluoromethylphenyl)guanidine, andN-(adamantan-1-yl)-N'-(2-trifluoromethyl-4-fluorophenyl)guanidine; or acorresponding compound bearing 1, 2, 3 or more additional or othersubstituents on one or both hydrocarbon groups, e.g., alkyl of 1-8carbon atoms, e.g., methyl-, ethyl; halo, e.g., chloro, bromo, iodo,fluoro; nitro; azido; cyano; isocyanato; amino; lower-alkylamino;di-lower-alkylamino; trifluoromethyl; alkoxy of 1-8 carbon atoms, e.g.,methoxy, ethoxy and propoxy; acyloxy, e.g., alkanoyloxy of 1-8 carbonatoms, e.g., acetoxy and benzoyl; amido, e.g., acetamido,N-ethylacetamido; carbamido, e.g., carbamyl, N-methylcarbamyl,N,N'-dimethyl carbamyl; etc.

N,N'-disubstituted guanidines, e.g., of Formulae I, III and IV, can actin an agonistic, antagonistic or inverse agonistic manner in relation tothe prototypical sigma benzomorphans. Those which act as antagonists cantherefore be expected to affect pupil size, heart rate and mentation ina direction opposite that caused by benzomorphans which can bedetermined by standard tests in laboratory animals. The type and levelof activity for a given dosage of each compound can be conventionallydetermined by routine experimentation using well known pharmacologicalprotocols for each of the activities; the corresponding indicationstreatable at that dosage will be well known to skilled workers based onthe pharmacological results. The compounds of this invention areparticularly noteworthy for their antipsychotic activity to treatpsychotic conditions, e.g., schizophrenia, by analogy to the knownagents prolixin and haloperidol and for diagnosing sigma receptorintoxicated conditions.

The invention is also related to the discovery that N,N'-disubstitutedguanidines having a high affinity for the sigma receptor are anxiolytic,and at the same time, are substantially non-sedative in an animal model.The term "high affinity to sigma receptor" means the compound exhibitsan IC₅₀ of less than 100 nM in a sigma receptor binding assay,preferably against ³ H--DTG as disclosed in the examples, below.Alternatively, the compounds may be tested against (+)-[³ H]3-PPP asdescribed by Largent, B. L., et al., Mol. Pharmacol. 32:772-784 (1987);Largent B. L., et al., Eur. J. Pharmacol. 155:345-347 (1988); andWikstrom, H., et al., J. Med. Chem. 30:2169-2174 (1987). The values ofIC₅₀ obtained by screening against ³ H--DTG and (+)-[³ H]3-PPP are wellcorrelated. See Weber, E. et al., Proc. Natl. Acad. Sci. USA 83:8784-8788 (1986).

These N,N'-disubstituted guanidines exhibit anxiolytic activities ofgenerally 100-1000 times greater than that of benzodiazepines. However,unlike benzodiazepines, the N,N'-disubstituted guanidines employed inthis invention are non-sedative. Therefore, these N,N'-disubstitutedguanidines are particularly useful for the treatment or prevention ofanxiety in animals.

Recent work by the inventors has shown that sigma receptor active drugsincluding the diaryl-guanidines can block acetylcholine release inducedby serotonin acting at 5HT₃ receptors in the guinea pig ileum myentericplexus (Campbell et al., J. Neurosci. 9:3380-3391 (1989)). The sigmareceptor active drugs act in a non-competitive manner to blockacetylcholine release stimulated by 5HT₃ receptor activation. Work byothers has shown that compounds acting as competitive antagonists at5HT₃ receptors have anxiolytic activity (Jones et al., Br. J. Pharmacol.93:985-993 (1988)). Therefore, the inventors reasoned thatnon-competitive antagonists of 5HT₃ receptor-induced acetylcholinerelease might also be anxiolytic. This invention shows that certainsigma receptor active N,N'-disubstituted guanidines indeed have potentanxiolytic activity.

Disubstituted guanidines are the subject of U.S. Pat. No. 4,709,094,whose disclosure is incorporated herein by reference. As a class, thesecompounds are described in this patent as exhibiting a highly selectivebinding activity to the sigma brain receptor.

Certain specific members of this class of disubstituted guanidines,i.e., those demonstrating a high affinity for the sigma receptor, areuseful for the treatment or prophylaxis of anxiety in an individualsusceptible to anxiety. Individuals susceptible to anxiety are those whohave experienced a plurality of prior episodes of GAD.

The anxiolytic activity of any particular N,N'-disubstituted guanidinemay be determined by use of any of the recognized animal models foranxiety. A preferred model is described by Jones, B. J. et al., Br. J.Pharmacol. 93:985-993 (1988). This model involves administering thecompound in question to mice which have a high basal level of anxiety.The test is based on the finding that such mice find it aversive whentaken from a dark home environment in a dark testing room and placed inan area which is painted white and brightly it. The test box has twocompartments, one white and brightly illuminated and one black andnon-illuminated. The mouse has access to both compartments via anopening at floor level in the divider between the two compartments. Themice are placed in the center of the brightly illuminated area. Afterlocating the opening to the dark area, the mice are free to pass backand forth between the two compartments. Control mice tend to spend alarger proportion of time in the dark compartment. When given ananxiolytic agent, the mice spend more time exploring the more novelbrightly lit compartment and exhibit a delayed latency to move to thedark compartment. Moreover, the mice treated with the anxiolytic agentexhibit more behavior in the white compartment, as measured byexploratory rearings and line crossings. Since the mice can habituate tothe test situation, naive mice should always be used in the test. Fiveparameters may be measured: the latency to entry into the darkcompartment, the time spent in each area, the number of transitionsbetween compartments, the number of lines crossed in each compartment,and the number of rears in each compartment. As disclosed more fullybelow in the examples, the administration of several N,N'-disubstitutedguanidines has been found to result in the mice spending more time inthe larger, brightly lit area of the test chamber. Unlike diazepam, theN,N'-disubstituted guanidines did not cause significant decreases in thenumbers of line crossings and rears. Thus, these N,N'-disubstitutedguanidines exhibit potent anxiolytic activity, and at the same time, arenon-sedating.

In the light/dark exploration model, the anxiolytic activity of aputative agent can be identified by the increase of the numbers of linecrossings and rears in the light compartment at the expense of thenumbers of line crossings and rears in the dark compartment, incomparison with control mice.

A second preferred animal model is the rat social interaction testdescribed by Jones, B. J. et al., supra, wherein the time that two micespend in social interaction is quantified. The anxiolytic activity of aputative agent can be identified by the increase in the time that pairsof male rats spend in active social interaction (90% of the behaviorsare investigatory in nature). Both the familiarity and the light levelof the test arena may be manipulated. Undrugged rats show the highestlevel of social interaction when the test arena is familiar and is litby low light. Social interaction declines if the arena is unfamiliar tothe rats or is lit by bright light. Anxiolytic agents prevent thisdecline. The overall level of motor activity may also be measured toallow detection of drug effects specific to social behaviors.

As noted above, the compounds of this invention are useful asanti-hypertensive agents and can be used in the same manner as knownantihypertensive agents, e.g., methyldopa, metoprolol tartrate andhydralazine hydrochloride.

Like guanidines generally and N,N'-diphenyl-guanidine specifically, thedisubstituted guanidines of this invention, including those of FormulaeI, III and IV, are accelerators for the vulcanization of rubbers, e.g.,natural rubbers and epoxy group-containing acrylic rubber, and can beused for such purpose in the same manner as N,N'-diphenylguanidine. Thus[³ H]--DTG can be incorporated into a vulcanized rubber object, e.g., atire tread, and rate of loss of rubber therefrom by water can bemonitored by rate of loss of radioactivity.

The N,N'-disubstituted guanidines can readily be prepared byconventional chemical reactions, e.g., when R and R' are the same, byreaction of the corresponding amine with cyanogen bromide. Other methodswhich can be employed include the reaction of an amine or amine saltwith a preformed alkyl or aryl cyanamide. See Safer, S. R., et al., J.Org. Chem. 13:924 (1948). This is the method of choice for producingN,N'-disubstituted guanidines in which the substituents are notidentical. For a recent synthesis of unsymmetrical guanidines, see G. J.Durant et al., J. Med. Chem. 28:1414 (1985), and C. A. Maryanoff et al.,J. Org. Chem. 51:1882 (1986).

Included as well in the present invention are the novel compoundsdisclosed herein as well as pharmaceutical compositions thereofcomprising an effective mount of the N,N'-disubstituted guanidine incombination with a pharmaceutically acceptable carrier.

The N,N'-disubstituted guanidines and the pharmaceutical compositions ofthe present invention may be administered by any means that achievetheir intended purpose. For example, administration may be by parenteralsubcutaneous, intravenous, intramuscular, intra-peritoneal, transdermal,or buccal routes. Alternatively, or concurrently, administration may beby the oral route. The dosage administered will be dependent upon theage, health, and weight of the recipient, kind of concurrent treatment,if any, frequency of treatment, and the nature of the effect desired.

Compositions within the scope of this invention include all compositionswherein the N,N'-disubstituted guanidine is contained in an amount whichis effective to achieve its intended purpose. While individual needsvary, determination of optimal ranges of effective mounts of eachcomponent is with the skill of the art. Typically, the compounds may beadministered to mammals, e.g. humans, orally at a dose of 0.0025 to 15mg/kg, or an equivalent amount of the pharmaceutically acceptable saltthereof, per day of the body weight of the mammal being treated forpsychosis, depression, hypertension, or anxiety disorders, e.g.,generalized anxiety disorder, phobic disorders, obsessional compulsivedisorder, panic disorder, and post traumatic stress disorders.Preferably, about 0.01 to about 10 mg/kg is orally administered to treator prevent such disorders. For intramuscular injection, the dose isgenerally about one-half of the oral dose. For example, for treatment orprevention of psychosis or anxiety, a suitable intramuscular dose wouldbe about 0.0025 to about 15 mg/kg, and most preferably, from about 0.01to about 10 mg/kg.

The unit oral dose may comprise from about 0.25 to about 400 mg,preferably about 0.25 to about 100 mg of the compound. The unit dose maybe administered one or more times daily as one or more tablets eachcontaining from about 0.10 to about 300, conveniently about 0.25 to 50mg of the anxiolytic compound or its solvates.

In addition to administering the compound as a raw chemical, thecompounds of the invention may be administered as part of apharmaceutical preparation containing suitable pharmaceuticallyacceptable carriers comprising excipients and auxiliaries whichfacilitate processing of the compounds into preparations which can beused pharmaceutically. Preferably, the preparations, particularly thosepreparations which can be administered orally and which can be used forthe preferred type of administration, such as tablets, dragees, andcapsules, and also preparations which can be administered rectally, suchas suppositories, as well as suitable solutions for administration byinjection or orally, contain from about 0.01 to 99 percent, preferablyfrom about 0.25 to 75 percent of active compound(s), together with theexcipient.

The pharmaceutical preparations of the present invention aremanufactured in a manner which is itself known, for example, by means ofconventional mixing, granulating, dragee-making, dissolving, orlyophilizing processes. Thus, pharmaceutical preparations for oral usecan be obtained by combining the active compounds with solid excipients,optionally grinding the resulting mixture and processing the mixture ofgranules, after adding suitable auxiliaries, if desired or necessary, toobtain tablets or dragee cores.

Suitable excipients are, in particular, fillers such as saccharides, forexample lactose or sucrose, mannitol or sorbitol, cellulose preparationsand/or calcium phosphates, for example tricalcium phosphate or calciumhydrogen phosphate, as well as binders such as starch paste, using, forexample, maize starch, wheat starch, rice starch, potato starch,gelatin, tragacanth, methyl cellulose, hydroxypropylmethylcellulose,sodium carboxymethylcellulose, and/or polyvinyl pyrrolidone. If desired,disintegrating agents may be added such as the above-mentioned starchesand also carboxymethyl-starch, cross-linked polyvinyl pyrrolidone, agar,or alginic acid or a salt thereof, such as sodium alginate. Auxiliariesare, above all, flow-regulating agents and lubricants, for example,silica, talc, steric acid or salts thereof, such as magnesium stearateor calcium stearate, and/or polyethylene glycol. Dragee cores areprovided with suitable coatings which, if desired, are resistant togastric juices. For this purpose, concentrated saccharide solutions maybe used, which may optionally contain gum arabic, talc, polyvinylpyrrolidone, polyethylene glycol and/or titanium dioxide, lacquersolutions and suitable organic solvents or solvent mixtures. In order toproduce coatings resistant to gastric juices, solutions of suitablecellulose preparations such as acetylcellulose phthalate orhydroxypropymethylcellulose phthalate, are used. Dye stuffs or pigmentsmay be added to the tablets or dragee coatings, for example, foridentification or in order to characterize combinations of activecompound doses.

Other pharmaceutical preparations which can be used orally includepush-fit capsules made of gelatin, as well as soft, sealed capsules madeof gelatin and a plasticizer such as glycerol or sorbitol. The push-fitcapsules can contain the active compounds in the form of granules whichmay be mixed with fillers such as lactose, binders such as starches,and/or lubricants such as talc or magnesium stearate and, optionally,stabilizers. In soft capsules, the active compounds are preferablydissolved or suspended in suitable liquids, such as fatty oils, orliquid paraffin. In addition, stabilizers may be added.

Possible pharmaceutical preparations which can be used rectally include,for example, suppositories, which consist of a combination of the activecompounds with a suppository base. Suitable suppository bases are, forexample, natural or synthetic triglycerides, or paraffin hydrocarbons.In addition, it is also possible to use gelatin rectal capsules whichconsist of a combination of the active compounds with a base. Possiblebase materials include, for example, liquid triglycerides, polyethyleneglycols, or paraffin hydrocarbons.

Suitable formulations for parenteral administration include aqueoussolutions of the active compounds in water-soluble form, for example,water-soluble salts. In addition, suspensions of the active compounds asappropriate oily injection suspensions may be administered. Suitablelipophilic solvents or vehicles include fatty oils, for example, sesameoil, or synthetic fatty acid esters, for example, ethyl oleate ortriglycerides. Aqueous injection suspensions may contain substanceswhich increase the viscosity of the suspension include, for example,sodium carboxymethyl cellulose, sorbitol, and/or dextran. Optionally,the suspension may also contain stabilizers.

The characterization of sigma receptors in vitro has been difficultbecause of the lack of selective drug ligands. Most benzomorphan opiatescross-react with other (mu, delta, kappa), opioid receptors and aretherefore of only limited value for characterizing and isolatingreceptors. Pasternak et al., J. Pharmacol. Exp. Ther. 219:192-198(1981); Zukin, R. S., et al., Mol. Pharm. 20:246-254 (1981); and Tam, S.W., Eur. J. Pharmacol. 109:33-41 (1985). [³ H]DTG binds specifically andwith high affinity to a single class of binding sites in guinea pigbrain membranes. The binding characteristics and the drug specificityprofile of these sites are concordant with those proposed for the sigmareceptor, including 1) naloxone insensitivity and stereo-selectivity fordextrorotatory isomers of benzomorphan opiates such as (+)SKF 10,047,(+)cyclazocine and (+)pentazocine; 2) high affinity for haloperidol andcertain phenothiazine antipsychotic drugs; 3) stereo-selectivity for(-)butaclamol; and 4) insensitivity to dopamine and apomorphine. [³H]--DTG is one of only two known tritiated compounds that are selectivefor the sigma site. The other, (+)[³ H]3-PPP, originally proposed to bea dopamine autoreceptor agonist, has recently been shown to be selectivefor sigma sites in rat brain membrane binding assays. Largent et al.(1984), supra. Our experiments confirm these findings in the guinea pigand show that [³ H]DTG and (+)[³ H]3-PPP have virtually identicalreceptor binding characteristics and drug selectivity profiles. Previousstudies have shown that sigma sites can also be labeled with (+)[³ H]SKF10,047, (+)[³ H]-ethylketazocine and with (±)[³ H]SKF 10,047. However,these ligands are not selective for the sigma site and require thepresence of appropriate drugs in the binding assays to maskcross-reacting non-sigma binding sites.

[³ H]DTG has a number of advantages as a sigma ligand. It is highlyselective for the sigma site (unlike [³ H]SKF 10,047 and (+)[³H]ethylketazocine), it has a high degree of specific binding (90-97% oftotal binding) and it has a relatively simple chemical structure that isnot chiral (unlike (+)[³ H]3-PPP and the benzomorphan opiates). Thesecharacteristics make it a good starting compound for the synthesis ofanalogs for structure-activity studies and for the design ofirreversible (after photolysis) sigma receptor ligands, e.g., compoundsof Formulae I and II wherein at least one of R and R' isazido-substituted carbocyclic aryl.

The sigma site labeled with [³ H]DTG is clearly not related toconventional (mu, delta, kappa) opioid receptors as it is naloxoneinsensitive and shows stereoselectivity for dextrorotatory isomers ofbenzomorphan drugs. This is a reversed stereoselectivity compared tonaloxone-sensitive opioid receptors which are selective for levorotatoryisomers of opiates. Sigma receptors should therefore not be referred toas sigma "opioid" receptors. The drug selectivity of sigma sites fordextrorotatory isomers of psychotomimetic opiates does, however,correlate well with the pharmacological profile of dextrorotatory versuslevorotatory opiates in animal tests designed to differentiate betweenconventional opioid receptor activity and sigma (behavioral) activity ofbenzomorphan drugs. Cowan, A., Life Sci. 28:1559-1570 (1981); Brady, K.T., et al., Science 215:178-180 (1982); and Khazan, N., et al.,Neuropharmacol. 23:983-987 (1984).

Autoradiography studies using [³ H]DTG visualize the sigma site inslide-mounted rodent brain sections and confirm that sigma sites aredifferent from mu, delta, and kappa opioid receptors as the distributionof [³ H]DTG binding is rather distinct from the distribution of mu,delta, kappa opioid receptors. The anatomical distribution of [³ H]DTGbinding sites is, however, very similar if not identical to thedistribution of [³ H]3-PPP binding sites, further confirming that thetwo radioligands label identical binding sites. The high affinity of the[³ H]DTG binding site for haloperidol and for certain phenothiazineantipsychotics (TABLE I) which are also dopamine D₂ receptor antagonistsraises the question as to the relation of sigma receptors to dopamine D₂receptors. The results presented show that the [³ H]DTG site is clearlydistinct from dopamine D₂ receptors, because the autoradiographicdistribution of dopamine receptor is dissimilar and because dopamine,apomorphine and many other dopamine receptor ligands do not interactwith the [³ H]DTG binding site.

Furthermore the sigma site labeled with [³ H]DTG is stereoselective for(-)butaclamol which is a reversed stereoselectivity compared to thedopamine D₂ receptors which are stereoselective for (+)butaclamol.

The haloperidol-sensitive sigma site labeled with [³ H]DTG was found tohave a moderate affinity for the potent hallucinogen PCP in competitionexperiments. This is in agreement with findings by others who use (+)[³H]SKF 10,047, (±)[³ H]SKF 10,047 or (±)-[³ H]3-PPP to label sigma sites.In PCP receptor binding assays, however, [³ H]-PCP labeled predominantly(but not exclusively) a haloperidol-insensitive PCP binding site, termedPCP/sigma opiate receptor by Zukin and colleagues, Zukin et al. (1981,1986), supra, which is separate from the haloperidol-sensitive sigmasite labeled with [³ H]DTG or (+)[³ H]3-PPP. In contrast, [³ H]DTGappears to label exclusively the haloperidol sensitive sigma site, sinceall specific binding is displaceable by haloperidol and the anatomicaldistribution of [³ H]DTG binding is distinct from the distribution ofPCP receptors. Furthermore, unlabeled DTG is virtually inactive in a [³H]-PCP binding assay (S. William Tam, E. l. DuPont De Nemours & Co.,Wilmington, Del., personal communication). There is some controversy asto which of the two binding sites is responsible for causing thebehavioral effects of PCP and psychotomimetic benzomorphan opiates andwould therefore correspond to the sigma receptor postulated by Martin etal. (1976), supra. Zukin and his collaborators have argued that thebehavioral effects of both PCP and psychotomimetic benzomorphan opiatesare mediated by the haloperidol-insensitive PCP site, to whichbenzomorphan opiates bind with moderate affinity. Largent et al. (1986),supra, cited circumstantial evidence suggesting that it is equallylikely that the behavioral effects of both PCP and psychotomimeticopiates are mediated through the haloperidol sensitive sigma site. As [³H]DTG exclusively labels the haloperidol sensitive sigma site and doesnot interact significantly with the haloperidol-insensitive PCP site,behavioral studies using DTG or other substituted guanidines of thisinvention as prototypical sigma ligands, taking into account of whetherthey are agonists or antagonists (see below), should resolve this issue.

Perhaps the most important aspect of the findings on the drugspecificity of sigma sites that have emerged from this and other studiesis that they interact with certain very potent antipsychotic drugs(haloperidol, phenothiazines) that are used clinically to treatschizophrenia. This intriguing drug selectivity profile facilitatesstudies aimed at investigating the role of sigma receptors inantipsychotic drug action and abnormal brain function. The availabilityof DTG and like N,N'-disubstituted guanidines as a selective sigmaligand should serve to facilitate such studies.

The compounds of this invention have highly selective affinity for thesigma receptor. Consequently, they may have some of the activities ofthe benzomorphans, i.e., those produced by binding to thehaloperidol-sensitive sigma receptor but not those produced by thebinding of benzomorphans to other non-sigma receptors. For instance,benzomorphans may act at sigma receptors to cause mydriasis andtachycardia and pronounced psychotomimetic effects. DTG is therefore aneffective tool to demonstrate the physiological effects mediated by thesigma receptor which, to date, have been obscured by cross-reactivity ofbenzomorphans with non-sigma receptors.

The compounds of this invention are particularly valuable in thetreatment of humans afflicted with a psychotic disease, e.g.,schizophrenia, or with chronic hypertension. In this regard, they can beemployed in substantially the same manner as known antipsychotic agentsand anti-hypertensive agents, respectively.

Without further elaboration, it is believed that one skilled in the artcan, using the preceding description, utilize the present invention toits fullest extent. The following preferred specific embodiments are,therefore, to be construed as merely illustrative, and not limitative ofthe remainder of the disclosure in any way whatsoever. The entire textof all applications, patents and publications, if any, cited above andbelow are hereby incorporated by reference.

EXAMPLES

In the foregoing and in the following examples, all temperatures are setforth uncorrected in degrees Celsius and unless otherwise indicated, allparts and percentages are by weight.

Preparations

General Procedures. Melting points are uncorrected. N M R spectra wererecorded on a General Electric QE-300 spectrometer, operating at 300MHz. Chemical shifts (δ) are given in ppm using the residual protonsignal of the deuterated solvent as reference (CHD₂ OD δ3.300, CHCl₃δ7.260, HDO δ4.80), or the ¹³ C signal of the solvent (CD₃ OD δ49.00,CDCl₃ δ77.00). All solvents were reagent grade quality. Where drysolvents were needed, these were distilled from CaH₂ or Na before use.

Adamantan-1-ylcyanamide was prepared from the amine with cyanogenbromide in Et₂ O, as previously described by Geluk, N. W., et. al., J.Med. Chem. 12:712-716 (1969). (2-Methylphenyl)cyanamide was preparedsimilarly.

N,N'-Di-(adamantan-1-yl)guanidine hydrochloride was prepared accordingto the procedure of Geluk et al., supra.

Example 1 N,N'-Di-(4-bromo-2-methylphenyl)guanidine (4-Br--DTG)

To a stirred solution of cyanogen bromide (846 mg, 8.06 mmol) indistilled water (70 ml) was added in small portions 2.997 g (16.11 mmol)of 4-bromo-2-methylaniline (Aldrich, recrystallized from etherpentane).A white precipitate formed during the addition. The mixture was stirredat 80° C. for 4 h. Upon cooling at 0° C. for 12 h, a sticky yellow oilseparated out and was discarded. The clear aqueous phase wasconcentrated to about 30 ml. The white precipitate which formed wasredissolved by heating the mixture. This was then set aside at 4° C. for12 h. Filtration gave 430 mg of white solid. A 200-mg portion wasdissolved in 10 ml of hot water and treated with 5 ml of 10 KOHsolution. The mixture was extracted with CHCl₃ and the extract waswashed with brine and then dried (MgSO₄). Evaporation of the solventgave 171 mg of a brown solid which was crystallized from CHCl₃, giving120 mg (8%) 4-Br--DTG as small white needles: mp 209°-210°; NMR (300MHz, CD₃ OD, TMS) δ2.24 (s, 3), 7.12-7.25 (AB, 2, J=8 Hz), 7.35 (s, 1 );IR (KBr) 3460, 3340, 1630 cm⁻¹. Analysis calculated for C₁₅ H₁₅ N₃ Br₂ :C, 45.37; H, 3.81; N, 10.58. Found: C, 45.34; H, 3.56; N, 10.50.

Example 2 [³ H]-N,N'-di-ortho-tolyl-guanidine ([³ H]DTG)

Twenty-five mg (0.1 mmol) of the thus-produced 4-Br--DTG were submittedto Amersham Corporation (Arlington Heights, Ill.) for catalyticreduction in the presence of 20 Ci of [³ H]-gas. Two mCi portions of thecrude, radioactive product in 0.2 ml/each of 25% ethanol were purifiedby reverse phase high performance liquid chromatography (RP-HPu) on aVydac TP218 octadecasilica column using a CH₃ OH gradient (0-35% in 60minutes) in 0.1% trifluoroacetic acid for elution. How rate was 1ml/min. One minute fractions were collected. Aliquots of the fractionswere diluted 100 fold and 10 ul aliquots of the diluted fractionscorresponding to 0.1 ul of the original fractions were dissolved in 10ml scintillation fluid and counted in a scintillation spectrometer. Theequipment consisted of 2 Waters HPLC pumps, and automated electronicgradient controller and a Kratos variable wave length spectrophotometer.The radioactivity eluted as a major symmetrical peak coinciding with amajor, symmetrical UV (220 nm) absorbing peak at 41 minutes. This is thesame elution time at which authentic, unlabeled DTG emerges from thecolumn in this RP HPLC system. The specific activity of [³ H]DTG wasfound to be 52 Ci/mmol based on the amount of DTG under the major UVabsorbing peak as determined by quantitative UV-spectrophotometry andthe amount of radioactivity associated with this peak as determined byquantitative liquid scintillation spectrometry.

Following this procedure, or one of another conventional labellingsynthetic techniques well known in the art, the tritium labeled versionsof other N,N'-disubstituted guanidines of this invention, e.g., those ofTable I, can be produced. If only one of the N- and N'-groups employedas starting material bears a functional group convertible to an [³ H]bearing group, the resulting N,N'-disubstituted guanidine will be singlytagged. If both bear such a functional group, the resultingN,N'-disubstituted guanidine will be polytagged.

Example 3N-(2-Methyl-4-isothiocyanatophenyl-N'-(2-methylphenyl)-guanidine[Di-tolyl-Guanidine-Isothiocyanate (DIGIT)]

a. N-(2-Methyl-4-nitrophenyl)-N'-(2-methyl-phenyl)guanidineHydrochloride and the corresponding free base

A vigorously stirred mixture of 2-methylphenyl-cyanamide [1.107 g, 0.380mmol, prepared from o-toluidine by the method of Safter et al. (1948)]and 2-methyl-4-nitroaniline hydrochloride in chlorobenzene (45 ml) washeated at 90° for 3 h and then allowed to cool to 25°. The resultingpale yellow precipitate was collected, washed with CH₂ Cl₂, and dried,giving 2.284 g (91%) of the hydrochloride of the desired product (pureby NMR). A 681 mg sample was recrystallized twice from absolute EtOH togive 600 mg (88%) of the desired product as pale yellow microcrystalssuitable for the next reaction: mp 196-200; (CD₃ OD, 300 MHz) δ2.37 (s,3), 2.47 (s, 3), 7.33-7.40 (m, 4), 7.59 (d, 1), 8.18 (dd, 1), 8.27 (d,1). A 473 mg sample of the hydrochloride was dissolved in 50 ml of hotwater, filtered, cooled to 25°, and treated with 5 ml of 5N NaOH. Theresulting bright yellow precipitate of the title compound (free base)was dried (403 mg, 92%) and then recrystallized twice from 95% EtOH togive the analytical sample as yellow platelets: mp 177-179; (DC₃ OD, 300MHz) δ2.31 (s, 6), 7.01-7.35 (m, 5), 7.98 (dd, 1), 8.06 (d, 1). Analysiscalculated for C₁₅ H₁₆ N₄ O₂ ; C, 63.37; H, 5.67; N, 19.71. Found: C,63.41; H, 5.42; N, 19.90.

b. N-(2-Methyl-4-aminophenyl)-N'-(2-methylphenyl)-guanidinehydrochloride

A Parr hydrogenation flask was charged with a solution ofN-(2-methyl-4-nitrophenyl)-N'-2-(methylphenyl)-guanidine hydrochloride(478 mg) in 30 ml of absolute EtOH. 30% Pd on charcoal (71 mg) was addedand then the mixture was hydrogenated at 60 psi at 25° C. for 12 h. Allfurther operations were carried out under an atmosphere of Ar. Themixture was centrifuged, filtered through Celite and the filtrate wasconcentrated in vacuo to 2 ml. Ether (21 ml) was added and after 4 h at20°, the white precipitate was collected and dried, giving 406 mg (78%)of the title compound (amine hydrochloride) suitable for the nextreaction: mp 232.5°-234.5°, (CD₃ OD, 300 MHz) δ2.22 (s, 3), 2.34 (s, 3),6.60 (dd, 1), 6.66 (d, 1), 6.98 (d, 1), 7.26-7.38 (m, 4).

c. N-(2-Methyl-4-isothiocyanatophenyl)-N'-(2-methylphenyl)-guanidineHydrochloride (DIGIT)

NaOAc (1.767 g, 21.5 mmol) and HOAc (0.839 g, 14.0 mmol) were dissolvedin dry MeOH such that the final volume of the solution was 100 ml. A12.7 ml aliquot of this solution was added to 199 mg (0.686 mmol) ofN-(2-methyl-4-aminophenyl)-N'-(2-methylphenyl)guanidine hydrochlorideunder an Ar atmosphere. Thiophosgene (54.7 ul, 86.7 mg, 0.754 mmol,freshly distilled) was then injected into the stirred reaction mixture.The reaction was complete upon mixing as judged by silica gel TLC(500:100:1, CHCl₃ --MeOH--HOAc). Water (20 ml) was added and the mixturewas concentrated in vacuo to 19 ml.

The next steps were done in rapid succession in order to minimize thepossible reaction of the isothiocyanate group with the guanidine freebase grouping of another molecule. The above 19 ml concentrate wascooled to 0° and treated with ice-cold saturated NaHCO₃ (15 ml). Theresulting mixture containing a white precipitate was extracted withCHCl₃ (4×15 ml). The combined extracts were washed with ice cold brine,dried (MgSO₄), filtered through Celite, and cooled to 0°. Next, excessHCl gas was bubbled through the colorless solution and then the solutionwas concentrated in vacuo to 25 ml and hexane (20 ml) was added. Fouradditional times the mixture was concentrated again and more hexane wasadded. Evaporation of the final mixture to dryness gave crudehydrochloride as a gummy white solid (263 mg, 115%). This was taken upin absolute ethanol (2 ml), and diluted with ether (80 ml) to give acloudy white suspension from which small clusters of white needlesformed on standing. The crystals were centrifuged, washed with ether(3×5 ml), and dried, giving 173 mg (76%) of the title compound: m.p.195°-197° C. (preheated bath); (CD₃ OD), 300 MHz) δ2.347 (s, 3), 2.351(s, 3), 7.23 (d, 1), 7.31 (s, 1), 7.34 (d, 1), 7.26-7.40 (m, 4); IR(KBr) 3466, 3138, 2927, 2152, 2119, 1646, 1631 cm⁻¹. Analysis calculatedfor C₁₆ H₁₇ ClN₄ S: C, 57.74; H, 5.15; N, 16.83. Found: C, 57.60; H,5.20; N, 16.64.

A tri-tritiated version of DIGIT is prepared starting with tritiatedN-(2-methyl-4-aminophenyl)-N'-(2-methylphenyl)-guanidine, which wasprepared by catalytic tritiation (Amersham) ofN-(2-methyl-4-nitro-6-bromophenyl)-N'-(2-methyl-4,6-dibromophenyl)-guanidine.The tri-tritiated amino compound was also used as the immediateprecursor for the preparation of the tritritiated version of the 4-azidocompound the preparation of which is described hereinafter and which wasused in the solubilization of sigma receptors.

Example 4 N, Adamantan-1-yl-N'-cyclohexylguanidine HCl

Adamantan-1-ylcyanamide (514 mg, 2.92 mmol), and cyclohexylaminehydrochloride (398 mg, 2.93 mmol) were finely ground together, andheated at 200° C. for 10 min. The resultant glassy solid was pulverized,and extracted twice with 50 ml boiling 5% HCl. The insoluble materialwas filtered, and dried. 346 mg, mp 264°-270° (p.h.b. 240° C.). Oncooling the combined aqueous extracts to 25°, a white ppt. formed, andwas filtered off, 56 mg. Combined crude yield: 44%. Crystallization of a50 mg sample of the crude product from EtOH/Et₂ O afforded white needles(25 mg, mp 269°-271° C. (p.h.b. 240°), lit. 268°-269° C.). ¹ H (CD₃ OD)δ1.208-1.474 (m, 5H), 1.744 (s, 9H), 1.967 (s, 8H), 2.119 (5, 3H), 3.434(m, 1H), ¹³ C (CD₃ OD) (broad band decoupled) 25.55, 26.26, 30.96,33.74, 36.74, 42.73.

Example 5 N-Cyclohexyl-N'-(2-methylphenyl)guanidine

A suspension of cyclohexylamine hydrochloride (310 mg, 2.28 mmol) and(2-methylphenyl)cyanamide (365 mg, 2.76 mmol) in dry chlorobenzene washeated at 125°-135° for 4 hrs. The solvent was evaporated in vacuo withheating, and the residue partitioned between CH₂ Cl₂ and 20×8 ml 10%HCl. The aqueous extracts were made alkaline to pH 9-9.5, and the whiteprecipitate collected after standing at 4° C. overnight. Tworecrystallizations from EtOH/H₂ O gave 140 mg (22%) of white needles, mp145°-146°, 1H (CD₃ OD) δ1.149-2.054 (m, 10H), 2.164 (s, 3H), 3.50 (m,1H), 6.875 (d, 1H, J=7.5 Hz), 6.968 (t, 1H, J=7.5 Hz), 7.118 (t, 1H,J=7.5 Hz), 7.177 (d, 1H, J=7.5 Hz). Anal. Calcd for C₁₄ H₂₁ N₃ : C,72.69; H, 9.15; N, 18.16. Found: C, 72.72; H, 9.24; N, 18.18.

Example 6 N-(Adamantan-1-yl)-N'-(2-methylphenyl)guanidine

A suspension of adamantan-1-ylcyanamide (151 mg, 0.857 mmol) ando-toluidine hydrochloride (186 mg, 0.857 mmol) in dry chlorobenzene washeated between 100°-130° C. for 4.5 hrs. The chlorobenzene wasevaporated in vacuo with heating and the residue (285 mg) taken up in 18ml of water. A gummy, insoluble material was discarded. On adjusting theaqueous extract to pH 9-9.5, a precipitate formed (194 mg, 71%). Tworecrystallizations from EtOH/H₂ O gave the analytical sample; mp160°-161°, ¹ H (CD₃ OD) δ1.742 (s, 6H), 2.050 (d, 6H, J=2.4 Hz), 2.029(s, 3H), 6.956 (j, 1H, J=8.1 Hz), 7.056 (t, 1H, J=8.1 Hz), 7.168 (t, 1H,J=7.5 Hz), 7.213 (d, 1H, J=7.5 Hz). Anal. Calcd for C₁₈ H₂₅ N₃ : C,76.28; H, 8.89; N, 14.83. Found: C, 76.25; H, 8.86; N, 14.60.

Example 7 N-(Adamantan-1-yl)-N'-(2-iodophenyl)guanidine HCl

Adamantan-1-ylcyanamide (299 mg, 1.70 mmol) and o-iodoanilinehydrochloride (433 mg 1.70 mmol) were finely ground together and heatedat 200° for 10 min. The resultant black glassy solid was pulverized, andrecrystallized 5 times from EtOH/Et₂ O. The resultant faintly blueneedles (80 mg) were dissolved in 4 ml EtOH, and passed through a short(<1 cm) column packed from bottom to top with Celite, charcoal,activated alumina, and sand. The colorless eluate was then diluted with5 ml Et₂ O, and allowed to stand in an Et₂ O diffusion chamberovernight. The white needles were collected by suction filtration, 40mg, mp 274°-275° C. Addition of another 5 ml Et₂ O yielded a second crop(31 mg) of identical material. Combined yield: 21%. ¹ H (CD₃ OD) δ1.779(s, 6H), 2.090 (s, 6H), 2.164 (s, 3H), 7.168 (t, 1H, J=8.1 Hz), 7.388(d, 1H, J=9.1 Hz), 7.503 (t, 1H, J=7.8 Hz), 8.004 (d, 1H, J=7.8 Hz).

Example 8 N-(2-Methyl-4-azido-phenyl)-N'-(2-methylphenyl)guanidine

N-(2-Methylphenyl)-N'-(2-methyl-4-aminophenyl)guanidine dihydrochloride(112 mg, 0.341 mmol) were dissolved in 2 ml H₂ O and 100 ul cone. HCl(1.2 mmol). The solution was cooled in an ice bath, and a solution ofNaNO₂ (42 mg, 0.61 mmol) in 450 ul H₂ O were added. The reaction mixtureturned yellow, and was stirred for 45 min before solid NaN₃ (43 mg,0.661 mmol) was added in a single portion. After N₂ evolution wascaused, a foamy solid (11 mg) was removed and discarded. Solid NaOH (96mg, 2.41 mmol) was added, and the bright yellow precipitate wasextracted with Et₂ O (3×5 ml). Evaporation of the combined Et₂ O layersgave a yellow solid which was crystallized from EtOH/H₂ O: NMR (CD₃ OD)δ2.279 (s, 3H), 2.284 (s, 3H), 6.864 (dd, 1H, J=2.4 Hz, 8.4 Hz), 6.914(d, 1H, J=2.1 Hz), 7.055 (td, 1H, J=1.8 Hz, 7.2 Hz), 7.136-7.229 (m,4H).

Example 9N-(2-Methyl-4-nitro-6-bromophenyl)-N'-(2-methyl-4,6-dibromophenyl)guanidine

N-(2-Methyl-4-nitrophenyl)-N'-(2-methylphenyl)guanidine, as the freebase (281 mg, 0.987 mmol) was dissolved in 4 ml MeOH, and cooled in anice-bath. N-bromosuccinimide (freshly recrystallized from H₂ O) (531 mg,2.98 mmol) was added in two portions over 15 min. After 1.5 hrs thebrown sludgy reaction mixture was diluted with 4 ml MeOH, and allowed towarm to 25° C. A brown solid was filtered off (266 mg), and crystallizedfrom acetone/H₂ O, to afford brown needles (2.6 mg, 42%, mp 193°-195°C.). Sublimation of a 56 mg sample of these crystals at 0.01 mm Hg and170° afforded the analytical sample as a bright yellow amorphous solid(38 mg, mp 210°-213° C.). NMR (CD₃ OD) δ2.357 (s, 3H), 2.488 (s, 3H),7.444 (d, 1H, J=1.5 Hz), 7.669 (d, 1H, J=1.8 Hz), 8.033 (d, 1H, 2.1 Hz),8.267 (d, 1H, 2.4 Hz). Anal. Calcd for C₁₅ N₁₃ Br₃ N₄ O₂ : C, 34.58; N,2.52; N, 10.75. Found: C, 34.64; N, 2.41; N, 10.65.

Example 10 N,N'-Bis(2-iodophenyl)guanidine

A solution of cyanogen bromide (4.4042 g, 38.2 mmol) and 2-iodoaniline(4.138 g, 18.9 mmol) in H₂ O (70 ml) was heated at 70°-80° C. for 5 h.The reaction mixture was decanted from an off-white solid (1.90 g) whichwas discarded, and the supernatant was heated at the same temperature anadditional 16 h. On cooling to 25° C., the title compound precipitatedfrom solution as its hydrobromide salt and was centrifuged off, anddried (500 mg, 10%). This white powder was dissolved in boiling H₂ O (20ml), and 5N NaOH (2 ml) was added to the clear solution. The resultingwhite precipitate (290 mg) was washed with H₂ O (3×4 ml), andcrystallized from 95% EtOH, to give the title compound (119 mg, 39% fromthe hydrobromide salt) as long white needles: mp 161°-162° C. Onefurther crystallization provided the analytical sample: mp 161°-162° C.Anal. Calcd for C₁₃ N₁₁ N₃ I₂ : C, 33.72; N, 2.39; N, 9.07. Found: C,33.80; N, 2.26; N, 8.78. ¹ N NMR: δ6.790 (t, J=7.8 Hz, 2H), 7.304 (t,3=7.8 Hz, 2H), 7.506 (d, 3=7.8 Hz, 2H), 7.817 (d, 3=7.8 Hz, 2H). IR:729, 753, 1467, 1502, 1572, 1613, 1647, 3056, 3387 cm⁻¹.

Example 11 N,N'-Bis(3-methylphenyl)guanidine

Cyanogen bromide (788 mg, 7.44 mmol) was placed in a 25 ml round bottomflask, and m-toluidine (1.89 g, 17.6 mmol) was added dropwise. After theexothermic reaction had subsided, the residue was taken up in CH₂ Cl₂(20 ml), and was extracted with 5% HCl (5×10 ml). The aqueous extractswere adjusted to pH 10 with 6N NaOH. The resulting precipitate (674 mg,38%) was filtered off and crystallized from EtOH/H₂ O to give the titlecompound (240 mg, 14%) as white needles: mp 105°-106° C. ¹ H NMR: δ2.289(s, 6H), 6.814 (d, 2H, J=7.5 Hz), 6.939 (d, 2H, J=7.5 Hz), 6.981 (s,2H), 7.141 (t, 2H, J=7.5 Hz). Anal. Calcd for C₁₅ H₁₇ N₃ : C, 75.28; H,7.16; N, 17.56. Found: C, 75.42; H, 7.11; N, 17.43.

References

¹ Geluk, H. W., et al., J. Med. Chem. 12:712 (1969).

² Kazarinova, N. F., et al., Zn. Anal. Khim. 28:1853 (1973); Chem.Abstr. 80:97021 (1973).

Example 12 Synthesis of N,N'-Di(o-tolyl)-2-imino-imidazolidine

a. Synthesis of N,N' Ditolyl oxalodiamide

Oxalyl chloride (32 mmol) in methylene chloride (16 mL) was addeddropwise to a solution of o-toluidine (67 mmol) in methylene chloride (4mL) over a period of 10 min at 4° C. After the exotherm subsided, thesolution was removed from the ice bath and stirred at ambienttemperature for 2 h. A white precipitate had formed. The precipitate wasfiltered off, dried, and found to weigh 724.6 mg (90%). The amide andthe hydrooxalate salts were partially dissolved in methylene chloride(20 mL) and extracted with 1N HCl (5×, 15 mL). The resultant whitesuspension was filtered to provide a white solid (61%, mp 103°-104° C.).¹ H NMR (CD₃ CN/DMSO): δ2.259 (6H, s), 7.128-7605 (8H, m). IR (KBr):1298.7, 1642.9 (amide).

b. Synthesis of N,N' Bistolyl ethylene diamine

The following procedure was adapted from H. C. Brown, J. Org. Chem.38:912 (1978). Diborane (5 mmol) in THF was added dropwise to a THFsolution of N,N' Ditolyl oxalodiamide (443.0 mg, 1.65 mmol) over 10 minat 0° C. After 30 min, the reaction mixture was allowed to stir atambient temperature for 6 h. Then the solution was refluxed for 3 h. Theresulting yellowish solution was allowed to cool to 25° C. The solutionwas then acidified dropwise with 15% HCl (15 mL) via an additionalfunnel over 20 min. Gas evolution was noted and a white precipitate hadformed. The THF was then distilled off from the water throughrotoevaporation at 25° C. The water was then made basic with an excessof NaOH and then extracted with ether. The ether layer was washed withbrine, then dried over anhydrous potassium carbonate. The ether layerwas then concentrated to dryness to provide a tannish brown liquid. Theliquid was immediately taken up again in dry ether. Following thisprocedure, the solution was made acidic by adding ethereal HCl (10 mL)dropwise. A white solid had formed. This solid was immediately filteredoff and dissolved in ethanol (5 mL) and placed into an ether diffusionchamber. After 2 days, white prisms were found (158.1 mg, 30.6%), mp268°-270° C.

IR (KBr): in comparison with IR of diamide the bands at 1643.8 and1298.7 cm⁻¹ had disappeared.

c. Synthesis of N,N' Bistolyl-2-imino-imidazolidine

N,N' Bistolyl ethylene diamine (105.8 mg, 0.44 mmol) was taken up inEtOH (3 mL) to provide a light purple solution. This solution was thenplaced in a one-necked round-bottom flask (25 mL) equipped with magneticstirbar and reflux condenser. To this solution, cyanogen bromide (50 mg,0.47 mmol) in ethanol (2 mL) was added in a single portion. Theresultant reaction mixture was stirred for 1 h. It was noted that thesolution turned clear. The solution was then brought to reflux andmaintained at that temperature for 16 h. The reflux condenser was thenremoved, allowing the solvent to evaporate. The reaction mixture wasthen fused at 150° C. for 30 min to provide a brown solid. This solidwas immediately taken up in ethanol (4 mL) and placed into a centrifugetube. To this solution 1N NaOH was added (8 mL) to provide a "whispy"tan precipitate. After several failed attempts to pellet theprecipitate, the solution was simply extracted with chloroform (20 mL).The chloroform was concentrated to dryness to provide a clear oil (108.1mg, 92.6%).

Example 13 Synthesis ofN-Cyclohexyl-N'-(2-methyl-4-bromophenyl)guanidine

A solution of bromine (0.336 g, 2.1 mmol) in glacial acetic acid (1 ml)was added dropwise to a stirred solution ofN-cyclohexyl-N'-(2-methylphenyl)guanidine (0.231 g, 1 mmol) in glacialacetic acid (2 ml) at room temperature. After the addition, the additionfunnel was replaced by a reflux condenser and the red reaction solutionwas heated on a warm water bath at 70° C. for about an hour. Thereaction mixture was allowed to cool to room temperature and then pouredin cold ice water (30 ml) containing a 200 mg of sodium bisulfite. Themixture was then extracted with dichloromethane (3×15 ml) and thecombined organic layers were dried over sodium sulfate, filtered andconcentrated to give a colorless liquid (0.475 g). This liquid waspurified on a flash silica gel column to give a bright foamy product(0.25 g), mp 89°-91° C.

¹ H NMR (CDCl₃): δ7.4 (s, 1H, Ar-3-H); 7.32 (dd, 1H, J=8.29 and 1.52 Hz,Ar-5-H); 6.99 (d, 1H, J=8.34 Hz, Ar-6-H); 2.22 (s, 3H--Ar--CH3) and1.95-1.11 (m, 11H, cyclohexyl).

Mass (CI): 310, 312 (M⁺), 232 (M⁺ --Br).

Example 14 Synthesis ofN-(Adamantan-1-yl)-N'-(2-trifluoromethyl-4-fluorophenyl)guanidine

1-Adamantylcyanamide--A 2 liter glass three neck round bottom flaskequipped with a mechanical stirrer, thermometer and 250 ml pressureequalizing addition funnel was charged with 1-adamantanamine (72.25 g,0.48 mol), anhydrous ethyl ether (1L) and the solution cooled in an icebath (0°-5° C.). The addition funnel was charged with a solution ofcyanogen bromide (327.0 g, 0.31 mol) in anhydrous ethyl ether (200 ml).The reaction apparatus was purged with N₂ and a N₂ atmosphere maintainedthroughout the reaction. The cyanogen bromide was added dropwise withstirring so as to maintain the temperature of the reaction mixture below5° C. The addition required 35 minutes and upon completion the ice bathwas removed and the reaction mixture allowed to reach room temperature.The reaction mixture was allowed to stir overnight. The reaction mixturewas filtered (sintered glass funnel) to remove the adamantylaminehydrobromide and the filtrate concentrated in vacuo (Buchi Rotovap RE111) at 10 torr (aspirator vacuum) to afford the crude product. Thecrude product was dissolved in hot ethanol (100 ml) and distilled water(200 ml, room temperature) was added or until the point where turbiditywas observed. The flask and contents were placed in the refrigeratorover night. The product crystals were collected by filtration and airdried briefly (15 min). The product was then dried in a large capacityAbderhalden drying pistol at room temperature at 0.1 mm for 48 h toafford 46.01 g of 1-adamantylcyanamide.

N-(Adamantan-1-yl)-N-(2-trifluoromethyl-4-fluorophenyl)guanidineHydrochloride. A single neck 50 ml round bottom flask was charged withchlorobenzene (14 ml), 2-trifluoromethyl-4-fluoroaniline hydrochloride(L. M. Weinstock et al., Tet. Lett. 1419 (1978)) (1.0 g, 4.64 mmol) and1-adamantylcyanamide (0.82 g, 4.64 mmol). The reaction apparatus waspurged with N₂, and the reaction heated at reflux under N₂, whereupon aclear colorless solution resulted. Within minutes, a thick whiteprecipitate had formed. After 4.5 hr, the heating mantle was removed andafter cooling to room temperature diethyl ether (30 ml) was added andthe white solid (1.3 g) was collected by filtration. A portion of thiswhite solid (0.6 g) was crystallized from iso-propanol and the resultingwhite crystals were filtered and dried in vacuo at 100° C. to give thetitle product (0.35 g), mp 260°-61° C.

Anal. Calcd for C₁₈ H₂₂ N₃ ClF₄ (391.83): C, 55.17; H, 5.66; N, 10.72.Found: C, 55.32; H, 5.58; N, 10.88.

Example 15 Synthesis ofN-(Adamantan-1-yl)-N'-(2-trifluoromethylphenyl)guanidine)

A single neck glass 100 ml round bottom flask was charged withchlorobenzene (25 ml), o-(trifluoromethyl)-aniline hydrochloride(prepared from the free base, Aldrich, by dissolving in 5% HCl in MeOHand concentrating, 5.93 g, 30.0 mmol) and 1-adamantylcyanamide (5.29 g,30.0 mmol).

The reaction apparatus was purged with N₂ and the reaction heated atreflux under N₂, whereupon a clear colorless solution resulted. Withinminutes a thick white precipitate had formed and an additional 15 ml ofchlorobenzene was added to facilitate more efficient stirring. After 3h, the heating mantle was removed and after cooling to room temperaturethe white solid was collected by filtration. This material was dissolvedin boiling EtOH, filtered to remove insolubles, and ether added to thefiltrate until turbid. The white solid which crystallized on standingwas collected by filtration and dried in vacuo at room temperature togive the title product mp 259°-260° C.

Anal. Calcd for C₁₈ H₂₃ N₃ ClF₃ (373.83): C, 57.82; H, 6.20; N, 11.24.Found: C, 57.67; H, 6.20; N, 11.46.

Example 16 Synthesis ofN-(Adamantan-1-yl)-N'-(2,4-difluorophenyl)guanidine

A single neck glass 100 ml round bottom flask was charged withchlorobenzene (20 ml), 2,4-difluoroaniline hydrochloride (prepared fromthe free base, Aldrich, by dissolving in 5% HCl in MeOH andconcentrating, 2.0 g, 12 mmol) and 1-adamantylcyanamide (2.13 g, 12mmol).

The reaction apparatus was purged with N₂, and the reaction heated atreflux under N₂, whereupon a clear colorless solution resulted. Withinminutes a thick white precipitate had formed. After 2 h, the heatingmantle was removed and after cooling to room temperature the white solidwas collected by filtration. This material was recrystallized two timesfrom ethanol and the resultant white solid dried in vacuo at roomtemperature to give the title compound mp 258°-260° C.

Anal. Calcd for C₁₇ H₂₂ N₃ ClF₂ (341.82): C, 59.73; H, 6.49; N, 12.29.Found: C, 59.62; H, 6.44; N, 12.23.

Example 17 Synthesis of N,N'-Bis (3-ethylphenyl)-2-imino-imidazolidine

N,N'-di-(3-ethylphenyl)oxalodiamide Oxalyl chloride (19 mmol) inmethylene chloride (16 ml) was added dropwise to a solution of3-ethylaniline (40 mmol) in methylene chloride (4 ml) over a period of10 minutes at 4° C. After the exotherm subsided, the solution wasremoved from the ice bath and stirred at 25° C. for 2 hours. Thereaction mixture was diluted with another 40 ml of CH₂ Cl₂. Themethylene chloride solution was washed by aqueous HCl (1N, 40 ml×2),aqueous NaOH (3%, 30 ml×1), and brine (20 ml), dried, and concentrated.The crude product was washed with CH₂ Cl₂ (10 ml) as well as aqueous HCl(1N, 5-8 ml), the the precipitate was collected by filtration and driedunder vacuum to provide a white solidN,N'-di(3-ethylphenyl)-oxalodiamide in 55% yield. Mp 131° C.; ¹ H NMR(60 MHz, CDCl₃): δ1.2 (t, 6H), 2.6 (q, 4H), 6.8-7.6 (m, 8H). IR (CH₂Cl₂)=1690, 1520 cm⁻¹.

N,N'-Bis-(3-ethylphenyl)ethylene diamine Diborane (27.4 mmol, 27.4 ml,1M) in THF was added dropwise to a THF solution ofN,N'-di-(3-ethylphenyl)oxalodiamide (6.76 mmol, 2 g) over 10 minutes at0° C. After 30 minutes, the reaction mixture was refluxed for 16 hours.The resulting yellowish solution was allowed to cool to 25° C. Thesolution was then acidified by adding aqueous HCl (15%, 15 ml) over 20minutes; gas evolution was noted and a white precipitate had formed. TheTHF was then distilled off from the water through rotoevaporation at 25°C. The water was then made basic with excess of NaOH and then extractedwith CH₂ Cl₂. The CH₂ Cl₂ layer was washed with brine, then dried overanhydrous potassium carbonate. The CH₂ Cl₂ layer was then concentratedto dryness to provide an oil in 66% yield. ¹ H NMR (60 MHz,, CDCl₃):δ1.2 (t, 6H), 2.5 (q, 4H), 3.2 (s, 4H), 6.3-7.2 (m, 8H). IR (CH₂Cl₂)=1420, 1600 cm⁻¹.

N,N'-Bis (3-ethylphenyl)-2-imino-imidazolidineN,N'-Bis(3-ethylphenyl)-ethylene diamine (0.4 g, 1.49 mmol) in EtOH (2ml) was placed in a one necked round bottom flask equipped with amagnetic stir bar and reflux condenser. To this solution, cyanogenbromide (174 mg, 1.64 mmol) was added in a single portion. The solutionwas then brought to reflux for 15 hours. After reaction, the mixture wasconcentrated and recrystallized in MeOH/ether to yield a white crystalin 54% yield. MP 155° C.; ¹ H NMR (300 MHz, CDCl₃): δ1.25 (t, J=7.6 Hz,6H), 2.72 (q, J=7.6 Hz, 4H), 4.36 (s, 4H), 7.21-7.39 (m, 8H). ¹³ C NMR(300 MHz, CDCl₃): 15.08, 28.47, 49.45, 122.40, 124.48, 128.62, 130.31,134.91, 147.00, 154.39. IR (CH₂ Cl₂)=1470, 1550 cm⁻¹. Elementalanalysis. Calcd: C, 58.76; H, 6.49; N, 10.82. Found: C, 59.14; H, 6.83;N, 10.85.

Example 18 Characteristics of [³ H]DTG binding to guinea pig brainmembranes

Synthesis [³ H]DTG resulted in a pure homogenous product of highspecific radioactivity (52 Ci/mmol). [³ H]DTG bound specifically,saturably, reversibly, and with high affinity to guinea pig brainmembrane. In a typical experiment with 0.9 nM [³ H]DTG (30,000 cpm, 50%counting efficiency) the total binding was 2700 cpm while thenonspecific binding in the presence of 10 uM DTG or 10 uM haloperidolwas 50-150 cpm. Routinely, a specific binding to 90-97% of total bindingwas observed. At room temperature the binding of [³ H]DTG reachedequilibrium after 60-90 minutes and it was fully reversible afteraddition of 10 uM unlabeled DTG. Specific binding was linear with tissueconcentration between 2-40 mg tissue (original wet brain weight perassay tube). Binding of radioactivity to the glass fiber filters in theabsence of membranes was 10-20 cpm. Boiling of membranes at 100° C. for10 minutes prior to assay almost completely (90%) abolished specific [³H]DTG binding as did treatment of the membranes with trypsin and pronase(0.01 mg/ml for 30 min at room temperature), indicating that proteincomponents are important for the receptors binding ability.

To determine the equilibrium saturation binding of [³ H]DTG to guineapig brain membranes, membranes prepared as described herein wereincubated with [³ H]DTG at various concentrations from 0.3 nM to 90 nMin 1 ml 50 nM Tris/HCl buffer, pH 7.4, for 120 minutes at roomtemperature. Values obtained were the mean of quadruplicatedeterminations.

A Scatchard analysis of the saturation data shows a linear Scatchardplot with an apparent K_(D) of 28 nM and a maximum number of bindingsites (Bmax) of 85 pmol/g brain tissue (original wet weight). Analysisof the binding data with the curve fitting program LIGAND, Munson, P.J., et al., Anal. Biochem. 107:220-239 showed high compatibility with aone site binding model.

Example 19 Radioligand Binding Assays

Frozen guinea pig brains (Pel-Freeze, Rogers, Ariz.) were homogenized in10 volumes (w/v) of 0.32M sucrose using a Polytron homogenizer. Thehomogenate was spun at 900×g for 10 minutes at 4° C. The supernatant wascollected, and spun at 22,000×g for 20 minutes at 4° C. The pellet wasresuspended in 10 volumes of 50 mM Tris/HCl buffer, pH 7.4, incubated at37° C. for 30 minutes and spun again at 22,000×g for 20 minutes at 4° C.The pellet was then resuspended in 10 volumes of 50 mM Tris/HCl buffer,pH 7.4 and 10 ml aliquots of this membrane suspension were stored frozenat -70° C. until used in the binding assay. No effects of prolongedstorage (>3 months) of the membranes at -70° C. on sigma receptor numberor affinity for [³ H]DTG binding were observed.

For radioreceptor assays aliquots of the frozen membrane suspension werethawed and diluted tenfold with 50 nM Tris/HCl buffer, pH 7.4. To 12×75mm polystyrene or glass test tubes were added 0.8 ml of membranesuspension, 0.1 ml [³ H]DTG or (+)[³ H]3-PPP for a final concentrationof 0.9 nM, and 0.1 ml of unlabeled drugs or buffer. The proteinconcentration in the 1 ml final incubation volume was 800 ug,corresponding to 32 mg of brain tissue (original wet weight).Nonspecific binding was defined as that remaining in the presence ofeither 10 uM DTG or haloperidol for both the [³ H]DTG and the (+)[³H]3-PPP binding. After incubation for 90 minutes at room temperature themembrane suspension was rapidly filtered under vacuum through WhatmanGF/B glass fiber filters using a Brandel 48 well cell harvester(Brandel, Gaithersburg, Md.). The filters were washed with 3×5 mlice-cold 50 nM Tris buffer (pH 7.4 at room temperature). The filterswere dissolved in 10 ml each of Cytoscint (Westchem Products, San Diego,Calif.) and radioactivity was measured by liquid scintillationspectrometry at a counting efficiency of 35-50%. Saturation data wereevaluated by Scatchard analysis using both the EBDA NcPherson, G. A.,Computer Programs Biomed. 17:107-114 (1983), and LIGAND Munson, P. J.,et al., Anal. Biochem. 107:220-239 (1980), data analysis programs on anIBM Personal Computer-AT. IC₅₀ values were determined by plottingdisplacement curves onto semilogarithmic graph paper followed byinterpolation or by computerized non-linear least squares curve fitting(Fischer, J. B. et al., J. Biol. Chem. 263:2808-2816 (1988).

Utilizing the radioligand binding assay described above, the sigmareceptor binding activity based on [³ H]DTG displacement activity (IC₅₀value) for the sixteen (16) N,N'-disubstituted guanidine compoundslisted in Table I below was determined. The IC₅₀ value for each compoundis reported in Table I.

                  TABLE I                                                         ______________________________________                                                                 IC.sub.50 vs                                         Compound                 [.sup.3 H]DTG (nM)                                   ______________________________________                                        N,N'-di-o-tolyl-guanidine                                                                              32 ± 1                                            N,N'-di-n-butyl-guanidine                                                                              750 ± 33                                          N,N'-diphenyl-guanidine  397 ± 21                                          N,N'-diadamantyl-guanidine                                                                             17 ± 3                                            N-adamantyl-N'-2-methylphenyl-guanidine                                                                 3 ± 1                                            N,N'-di(2-methyl-4-bromophenyl)guanidine                                                               37 ± 3                                            N-(2-iodophenyl)-N'-(2-methylphenyl)-                                                                  21 ± 1                                            guanidine                                                                     N-(2-methyl-4-nitrophenyl)-N'-(2-methyl-                                                               37 ± 5                                            phenyl)-guanidine                                                             N,N'-di-(2,6-dimethylphenyl)guanidine                                                                   90 ± 18                                          N-(2,6-dimethylphenyl)-N'-(2-methylphenyl)-                                                            70 ± 3                                            guanidine                                                                     N-(adamantyl)-N'-(cyclohexyl)guanidine                                                                 13 ± 2                                            N,N'-di(cyclohexyl)guanidine                                                                           71 ± 7                                            N-(2-iodophenyl)-N'-(adamantyl)guanidine                                                                5 ± 1                                            N-(2-methylphenyl)-N'-(cyclohexyl)guanidine                                                            17 ± 5                                            N-(2-methyl-4-azidophenyl)-N'-(2-methyl-                                                               20 ± 1                                            phenyl)-guanidine                                                             N-(2-methylphenyl)-N'-(4-amino-2-methyl-                                                               280 ± 14                                          phenyl)-guanidine                                                             ______________________________________                                    

Fourteen of these compounds were found to be potent ligands of sigmareceptors as determined by their ability to displace [³ H]DTG from sigmareceptors in guinea-pig brain homogenates, the most potent beingN-(2-methylphenyl)-N'-(adamantan-1-yl)guanidine with an IC₅₀ of 2.6±0.6nM (n=6).

In a second series of experiments, selected compounds were testedagainst [³ H]MK-801 and [³ H]DTG. The results depicted in Table II areas follows (nM):

                  TABLE II                                                        ______________________________________                                                     IC.sub.50 vs*                                                    Chemical name  [.sup.3 H]DTG                                                                             [.sup.3 H]MK-801                                   ______________________________________                                        N-(1-Naphthyl)-N'-                                                                           40.1 ±                                                                             7.3 (4) 209 ±                                                                             50 (4)                                  (2-iodophenyl)-guanidine                                                      N-(Cyclohexyl)-N'-(4-                                                                        4.67 ±                                                                             1.04 (4)                                                                              23860 ±                                                                           8540 (5)                                bromo-2-methylphenyl)-                                                        guanidine                                                                     N,N'-Di-(4-indanyl)-                                                                         28.5 ±                                                                             7.5 (3) 506 ±                                                                             99 (2)                                  guanidine                                                                     N-(Adamantan-1-yl)-N'-(2-                                                                    7.44 ±                                                                             0.50 (3)                                                                              23100 ±                                                                           13100 (2)                               trifluoromethyl-phenyl)-                                                      guanidine                                                                     N-(Adamantan-1-yl)-N'-(2-                                                                    22.6 ±                                                                             1.9 (4) not done                                       methylphenyl)-N'-methyl-                                                      guanidine                                                                     N-(Adamantan-1-yl)-N'-(6-                                                                    21.9 ±                                                                             0.7 (4) >10,000 (2)                                    coumarinyl)-guanidine                                                         N-(Adamantan-1-yl)-N'-(8-                                                                    43.9 ±                                                                             2.0 (4)   6,700 (1)                                    coumarinyl)-guanidine                                                         N-(Adamantan-1-yl)-N'-                                                                       8.92 ±                                                                             0.48 (4)                                                                              >10,000 (1)                                    (2,4-difluorophenyl)-                                                         guanidine                                                                     N-(Adamantan-1-yl)-N'-(2-                                                                    4.27 ±                                                                             0.45 (4)                                                                               31,900 (1)                                    trifluoromethyl-4-fluoro-                                                     phenyl)-guanidine                                                             ______________________________________                                         *[s.e.m. (n)                                                             

Example 20 Drug Specificity of [³ H]DTG Binding

Displacement experiments were performed with drugs that are consideredtypical sigma ligands, as well as with drugs considered to beprototypical ligands for other neurotransmitter, neuromodulator, anddrug receptors. The IC₅₀ vs [³ H]DTG and vs. [³ H]3-PPP results aregiven in Table III below. These experiments showed that the [³ H]DTGbinding site is stereoselective for dextrorotatory benzomorphan opiatesand for (-)butaclamol; does not significantly interact with drugs thathave high affinities for acetylcholine, benzodiazepine, GABA, nor withmu, delta, or kappa opioid receptors; has a high affinity forhaloperidol and several drugs belonging to the phenothiazine class ofantipsychotics (haloperidol had the highest displacement potency of alldrugs tested); and has a moderate affinity for several other classes ofpsychoactive drugs, which included several tricyclic antidepressants,PCP, and the kappa opioid receptor ligand U50, 488H.

                  TABLE III                                                       ______________________________________                                                     IC.sub.50 vs.                                                                              IC.sub.50 vs.                                                    [.sup.3 H]DTG (nM)                                                                         (+)[.sup.3 H]3-PPP (nM)                             Drug         (± SEM)   (± SEM)                                          ______________________________________                                        Haloperidol  5 ±   0.3     17 ± 1                                       DTG          28 ±  1       53 ± 9                                       Perphenazine 42 ±  10      21 ± 3                                       (+)Pentazocine                                                                             43 ±  2       8 ±  3                                       (-)Pentazocine                                                                             135 ± 3       81 ± 1                                       (±)Pentazocine                                                                          69 ±  1       ND                                              (+)3-PPP     76 ±  4       33 ± 12                                      (-)3-PPP     280 ± 21      235 ±                                                                              60                                      (+)Cyclazocine                                                                             365 ± 25      47 ± 12                                      (-)Cyclazocine                                                                             2,600 ±                                                                             210     1,000 ±                                                                            0                                       Spiperone    690 ± 21      ND                                              (-)Butaclamol                                                                              530 ± 49      183 ±                                                                              5                                       (+)Butaclamol                                                                              2,150 ±                                                                             250     2,100 ±                                                                            71                                      (+)SKF 10,047                                                                              625 ± 88      93 ± 5                                       (-)SKF 10,047                                                                              4,000 ±                                                                             566     2,850 ±                                                                            390                                     PCP          1,050 ±                                                                             106     1,000 ±                                                                            71                                      U50,488H     1,350 ±                                                                             106     ND                                              Trifluoperazine                                                                            345 ± 4       ND                                              Trifluopromazine                                                                           605 ± 67      ND                                              Chlorpromazine                                                                             1,475 ±                                                                             265     ND                                              Amitriptyline                                                                              300 ± 7       ND                                              Imipramine   520 ± 14      ND                                              Desipramine  4,000 ±                                                                             566     ND                                              Nortriptyline                                                                              2,000 ±                                                                             640     ND                                              Guanabenz    4,600 ±                                                                             283     ND                                              Clonidine    >10,000      ND                                                  Cocaine      >10,000      ND                                                  ______________________________________                                         *IC.sub.50 is the molar concentration of the drug needed to produce           halfmaximal displacement of [.sup.3 H]DTG from sigma receptors. This is a     direct measure of the sigma receptor binding potency of the drug. ND = no     determined.                                                              

The above IC₅₀ s represent the average from 2-4 separate experiments (intriplicate). The following compounds caused no significant displacementat a 10 uM concentration: scopolamine, 5-OH-tryptamine, diazepam,bicuculline, picrotoxin, hexamethonium, dopamine, apomorphine, GABA,gamma-guanidino butyric acid, morphine, DAGO, metorphamide, dynorphin A,[leu⁵ ] enkephalin, beta-endorphin, naloxone, guanidino acetic acid,creatine, creatinine, 1,1-dimethyl-4-guanidine, methyl-guanidine,beta-guanidino propionic acid and cimetidine.

Example 21 Drug specificity of [³ H]DTG binding compared to (+)-[³H]3-PPP binding

Comparing the drug specificity of [³ H]--DTG binding with that of (+)[³H]3-PPP in the guinea pig, it was found that (+)[³ H]3-PPP boundspecifically, saturably (linear Scatchard plot), reversely and with highaffinity to guinea pig brain membranes (K_(D) =30 nm, Bmax=80 pmol/gfresh brain weight). The drug specificity profile of the (+)[³ H]3-PPPbinding in the guinea pig (Table II) was found to be very similar tothat reported in the rat. Largent et al. (1984), supra. Moreover, thedrug specificity profiles of typical sigma receptor active drugs in the(+)[³ H]3-PPP and [³ H]--DTG binding assays were highly correlated(r=0.95; p≦0.00001) which is consistent with the two compounds labelingthe same sites.

Example 22 Autoradiography Studies

Male Sprague Dawley rats (200-250 g) and NIH guinea pigs (300-350 g)were sacrificed, their brains rapidly removed and processed for receptorautoradiography according to the method of Herkenham et al. J.Neuroscience 2:1129-1149 (1982).

Fifteen um thick slide-mounted brain sections were incubated for 45minutes in 50 nM Tris-HCl (pH 8.0, 22° C.) containing 1 mg/ml bovineserum albumin (BSA) and 2 nM [³ H]DTG. Adjacent sections were incubatedwith 10 uM haloperidol or 10 uM DTG to measure nonspecific binding.Incubations were terminated by 4×2 minute washes in 10 nM Tris-HCl (pH7.4, 4° C.) with 1 mg/ml BSA, rapidly dried under a stream of cool airand placed in x-ray cassettes with ³ H-sensitive film (³ H-ultrofilm,LKB). Films were developed 6-8 weeks later (D-19, Kodak).

Example 23 Autoradiographic visualization of [⁸ H]DTG binding

Receptor autoradiography studies on guinea pig and rat brain sectionsusing [³ H]DTG showed a low density of specific binding diffuselydistributed throughout the gray matter of the rat and guinea pig brain.Superimposed on this homogeneous binding patterns was a heterogeneousdistribution of enriched binding in limbic and sensorimotor structures.The pattern of binding was more distinct in the guinea pig than rat.Similar observations for (+)[³ H]3-PPP autoradiography have beenreported. Largent et al. (1986), supra. Thus, description of [³ H]DTGbinding was drawn primarily from the guinea pig. In the forebrain,limbic structures moderately to densely labeled by [³ H]DTG were thediagonal band of Broca, septum, hypothalamus (especially theparaventricular nucleus), anterodorsal thalamic nucleus and zonaincerta. Sensorimotor thalamic nuclei moderately to densely labeledincluded the thalamic taste relay and reticular nuclei. Other thalamicnuclei labeled were the paraventricular and habenular nuclei. Very densebinding was seen in the choroid plexus. In the cortex dense [³ H]DTGlabeling occupied layer III/IV of retrospenial piriform, and entorhinalcortices. The rest of the cortex contained a low level of homogeneousbinding. The hippocampal formation exhibited discrete binding in thepyramidal granular cell layers. Sensorimotor areas of the midbrain wereselectively labeled by [³ H]DTG. The oculomotor nucleus, and morecaudally, the trochlear nucleus were very densely labeled, and thesuperior colliculus and red nucleus had moderate levels of binding.Other midbrain nuclei labeled were the dorsal raphe, interpeduncularnucleus, central gray, and the substantia nigra, para compacta. Theselective labeling of the para compacta in the guinea pig contrastedwith the low to moderate density of labeling present throughout thesubstantia nigra of the rat. In addition, very dense binding was foundin the subcommissural organ. In the hindbrain the locus coeruleus wasthe most densely labeled nucleus. Sensorimotor nuclei enriched in [³H]DTG binding sites were the trigeminal motor nucleus, nucleus of thefacial nerve, nucleus of the solitary tract, dorsal motor nucleus of thevagus, and the hypoglossal nucleus. Moderate to dense binding was alsofound throughout the gray matter of the cerebellum, and in the pontinereticular nuclei.

Example 24 Drug Specificity of [³ H]AZ--DTG binding

The haloperidol-sensitive sigma receptor binds [³H](+)3-[3-hydroxyphenyl]-N-(1-propyl)piperidine([³ H](+)-3-PPP) and [³H]1,3-di-o-tolylguanidine ([³ H]DTG), with high affinity. In order toelucidate its structure, photoaffinity labeling of the sigma receptorfrom guinea pig brain was accomplished using a novel radioactivephotolabile derivative of DTG, [³ H]-m-azido-1,3-di-o-tolylguanidine([H]AZ--DTG). In the dark, [³ H]AZ--DTG binds reversibly to sigma sitesin brain membranes with high affinity (kd=28 nM). The drug specificityprofile of [³ H]AZ--DTG binding to brain membranes is identical to thatof the prototypical sigma ligands [³ H]DTG and [³ H](+)-3-PPP. Forphotoaffinity labeling, membrane suspensions containing proteaseinhibitors were preincubated in the dark with [³ H]AZ--DTG, thenfiltered and washed over Whatman GF/B glass fiber filters. The filterswere then irradiated with long-wavelength UV light for a 15 minuteperiod. Filter-bound proteins were solubilized with 50 mM Tris pH 7.4,0.1% sodium dodecyl sulfate. Solubilized proteins were subjected toSDS-polyacrylamide gel electrophoresis. Fluorography of the SDS-PAGEgels revealed that [³ H]AZ--DTG was selectively incorporated into a 29kD polypeptide. Labeling of this polypeptide was completely blocked bythe sigma ligands DTG, (+)-3-PPP, (+)pentazocine, and haloperidol at aconcentration of 10 uM, while labeling was unaffected by morphine,serotonin, dopamine, scopolamine, or GABA at the same concentration.These results represent the first estimate of the size of the bindingsubunit of the haloperidol-sensitive sigma receptor.

Example 25 Additional sigma receptor binding assays

Sigma receptor binding assays using guinea pig brain membranehomogenates and the radioligands [³ H]DTG and (+)[³ H]3-PPP were done aspreviously described (Weber et al., P.N.A.S. (USA) 83:8784-8788 (1986)).Briefly, frozen whole guinea-pig brains (Biotrol, Indianapolis, Ind.)were homogenized in 10 volumes (w/v) of ice-cold 320 mM sucrose using aBrinkman polytron. The homogenate was centrifuged at 1,000×g for 20minutes at 4° C. The supernatant was centrifuged at 20,000×g for 20minutes at 4° C. The resulting pellet was resuspended in 10 initialvolumes of 50 mM Tris/HCl buffer at pH 7.4 and centrifuged at 20,000×gfor 20 minutes at 4° C. The resulting pellet was resuspended in 5initial volumes ice-cold 50 mM Tris/Hcl (pH 0.4), and the final volumewas adjusted to yield a protein concentration of 3 mg/ml, as determinedby dye-binding protein assay (Biorad) using BSA as the standard.Aliquots of 20-ml were stored at -70° C. until used, with no detectableloss of binding.

For [³ H]DTG binding assays, 20-ml aliquots of the frozen membranesuspension were thawed and diluted 1:3 in 50 mM Tris/HCl (pH 7.4). To12×75 mm polystyrene test tubes were added 0.8 ml of diluted membranesuspension, 0.1 ml of [³ H]DTG (46 Ci/mmol; see Weber et al., P.N.A.S(USA) 83:8784-8788 (1986) or (+)[³ H]3-PPP (NEN, 98 Ci/mmol) to yield afinal concentration of 1.4 nM, and 0.1 ml of unlabelled drugs or buffer.The protein concentration in the 1-ml final incubation volume was 800ug/ml, corresponding to 32 mg of brain tissue (original wet weight) andto a tissue concentration within the linear range for specific binding.Non-specific binding was defined as that remaining in the presence of 10uM haloperidol. Specific binding constituted >90% of total [³ H]DTGbinding. Incubations were terminated after 90 minutes at roomtemperature by addition of 4 ml of ice-cold 50 mM Tris/HCl (pH 7.4) andrapid filtration of the membrane suspension through Whatman GF/Bglass-fiber filters under vacuum, using a 48-well cell harvester(Brandel, Gaithersburg, Md.). The filters were washed 2 times with 4 mlof 50 mM Tris/HCl (pH 7.4). Total filtration and washing time was lessthan 20 seconds. Each filter was dissolved in 10 ml Cytoscint (Westchem,San Diego, Calif.), and radioactivity was measured by liquidscintillation spectrometry at a counting efficiency of approximately50%. IC₅₀ values were determined by interpolation fromdisplacement-curve plots on semilogarithmic graph paper.

The IC₅₀ binding values (nM) are as follows: N,N-di(o-tolyl)guanidine(DTG, 32); N-(2-iodophenyl)-N'-(adamant-1-yl)guanidine (AdIpG, 6.2±0.7);N-(o-tolyl)-N'-(adamant-1-yl)guanidine (AdTG, 7.6±0.3);N,N'-di(adamant-1-yl)guanidine (DAG, 11.8±3.4);N-(cyclohexyl)-N'-(adamant-1-yl)guanidine (AdChG, 12.5±2.2);N-(o-tolyl)-N'-(cyclohexyl)guanidine (13.0±1.0);N,N'-di-(2,6-dimethylphenyl)guanidine (DXG, 90±18);N-(o-tolyl)-N'-(4-amino-2-methylphenyl)guanidine (NH₂ --DTG, 280±20);N,N'-di(phenyl)guanidine (DPG, 397±21);N-(o-tolyl)-N'-(o-iodophenyl)guanidine (IC₅₀ =21);N,N'-di-(p-bromo-o-methylphenyl)guanidine (IC₅₀ =37);N,N'-di-(m-n-propylphenyl)guanidine (IC₅₀ =36);N-(o-tolyl)-N'-(p-nitro-otolyl)guanidine (IC₅₀ =37);N,N'-di-(1-tetralinyl)guanidine (IC₅₀ =58);N-(o-tolyl)-N'-(o-xylyl)guanidine (IC₅₀ =70);N,N'-di-(cyclohexyl)guanidine (IC₅₀ =71);N-(3,5-dimethyl-1-adamantanyl)-N'-(o-tolyl)guanidine (IC₅₀ =15);N-(3,5-dimethyl-1-adamantanyl)-N'-(oiodophenyl)guanidine (IC₅₀ =16);N-(1-adamantyl)-N'-(o-nitrophenyl)guanidine (IC₅₀ =30);N,N'-di-(endo-2-norbornyl)guanidine (IC₅₀ =16);N-(exo-2-isobornyl)-N'-(o-iodophenyl)guanidine (IC₅₀ =18);N,N'-di-(exo-2-norbornyl)guanidine (IC₅₀ =22);N-(exo-2-isobornyl)-N'-(o-tolyl)guanidine (IC₅₀ =25);N-(o-iodophenyl)-N'-(t-butyl)guanidine (IC₅₀ =20);N,N'-dibenzylguanidine (IC₅₀ =90);N-(adamant-1-yl)-N'-(o-isopropylphenyl)guanidine (IC₅₀ =24);N-(adamant-2-yl)-N'-(p-iodophenyl)guanidine (IC₅₀ =2.7);N-(cyclohexyl)-N'-(p-bromo-o-tolyl)guanidine (IC₅₀ =5.5);N-(adamantan-2-yl)-N'-(o-iodophenyl)guanidine (IC₅₀ =5.2);2-imino-1,3H-dibenzo[d,f]-[1,3]-diazepine (Bridge-DPG, >10,000);N,N'-di(methyl)guanidine (DMG, >10,000); (+)-3-PPP (76±4); (-)-3-PPP(280±21); (+)-pentazocine (43±2); (-)-pentazocine (135±3);(-)-cyclazocine (2600±210); (-)-SKF10047 (4000±566); haloperidol(5±0.3); BMY 14802 (120±15); rimcazole (1400±100); tiospirone (233±52);perphenazine (42±10); chlorpromazine (1475±265); sulpiride (>10,000);TCP (1100±110); PCP (1050±106); and MK-801 (>10,000).

Example 26 Effect of Diazepam and N,N'-Disubstituted Guanidines onLight/Dark Exploration of Mice

The inventors wish to thank Brenda Costall, University of Bradford,Bradford, BD7 1DP, England, for screening the sigma receptor ligandsaccording to the procedure disclosed by her and others in Jones et al.,Br. J. Pharmacol. 93:985-993 (1988). According to this procedure, malealbino BKW mice, 25-30 g, were housed 10 to a cage and allowed freeaccess to food and water. They were kept on reversed light cycle withthe lights on between 22 h 00 min and 10 h 00 min.

The apparatus was an open-topped box, 45 cm long, 27 cm wide and 27 cmhigh, divided into a small (2/5) area and a large (3/5) area by apartition that extended 20 cm above the walls. There was a 7.5×7.5opening in the partition at floor level. The small compartment waspainted black and the large compartment white. The floor of each of thecompartments was marked into 9 cm squares. The white compartment wasilluminated by a 100 W tungsten bulb 17 cm above the box and the blackcompartment by a similarly placed 60 W red bulb. The laboratory wasilluminated with red light.

All tests were performed between 13 h 00 min and 18 h 00 min. Each mousewas tested by placing it in the center of the white area and allowing itto explore the novel environment for 5 min. Its behavior was recorded onvideotape and the behavioral analysis was performed subsequently fromthe recording. Five parameters were measured: the latency to entry intothe dark compartment, the time spent in each area, the number oftransitions between compartments, the number of lines crossed in eachcompartment and the number of rears in each compartment.

The N,N'-disubstituted guanidines, dissolved in distilled water, wereadministered subcutaneously 40 min before testing over the dosage rangeof 0.01 ug to 0.1 mg/kg (5 mice per dosage level). Diazepam (0.063-10mg/kg) was dissolved in the minimum quantity of polyethylene glycol,diluted to the appropriate volume with distilled water and administeredintraperitoneally to five mice for each dosage level. The results areshown in FIGS. 1-6 (The cross hatched area=light area; solidcolumns=dark area).

As shown in FIG. 1, diazepam dose-dependently increased the proportionof time the mice spent in the larger, lighted area of the test chamber.The numbers of line crossings and rears in the light compartmentincreased at the expense of those in the dark compartment. At thehighest dose of diazepam (10 mg/kg), the numbers of rears and linecrossings decreased significantly showing that the drug was markedlysedative. The latency to entering the dark compartment increased with apeak effect at 0.25 mg/kg (latency=30 sec) while controls showed alatency of 9 sec (S.E.M.s<12.1%, P<0.001).

As shown in FIG. 2, N,N'-di-(adamantan-1-yl)guanidine tended to increasethe proportion of time the mice spent in the larger, lighted area of thetest chamber, with a peak at 0.01 ug/kg. The numbers of line crossingsand rears in the light compartment increased at the expense of those inthe dark compartment. At the highest dose of N,N'-di-(adamantan-1-yl)guanidine (0.1 mg/kg), the numbers of rears and line crossings did notdecrease significantly showing that the drug was not sedative at thislevel. The latency to entering the dark compartment increased with apeak effect at 0.01 ug/kg (latency=20 sec) while controls showed alatency of 12 sec (P<0.05<0.001).

As shown in FIG. 3, N-(adamantan-1-yl)-N'-(2-methylphenyl)guanidine dosedependently increased the proportion of time the mice spent in thelarger, lighted area of the test chamber. The numbers of line crossingsand rears in the light compartment increased at the expense of those inthe dark compartment. At the highest dose ofN-(adamantan-1-yl)-N'-(2-methylphenyl)guanidine (0.1 mg/kg), the numbersof rears and line crossings did not decrease significantly showing thatthe drug was not sedative at this level. The latency to entering thedark compartment also increased in a dose-dependent manner.

As shown in FIG. 5, N-(adamantan-1-yl)-N'-o-iodophenyl-guanidine alsoincreased, in a dose-dependent manner, the proportion of time the micespent in the larger, lighted area of the test chamber. The numbers ofline crossings and rears in the light compartment increased at theexpense of those in the dark compartment. At the highest dose ofN-(adamantan-1-yl)-N'-o-iodophenyl guanidine (0.1 mg/kg), the numbers ofrears and line crossings did not decrease significantly showing that thedrug was not sedative at this level. The latency to entering the darkcompartment also increased in a dose-dependent manner.

For comparison, a compound with a relatively low sigma receptor affinitywas tested for anxiolytic activity. As shown in FIG. 6,N-(3,5-dimethyladamantan-1-yl)-N'-{[(E)-2-phenylethenyl]phenyl}guanidine(IC₅₀ =1,000 nM) generally did not increase the proportion of time themice spent in the larger, lighted area of the test chamber. The numbersof line crossings and rears in the light compartment tended to remainthe same except at the highest dosage. At the highest dose ofN-(3,5-dimethyladamantan-1-yl)-N'-{[(E)-2-phenylethenyl]phenyl}guanidine(0.1 mg/kg), the numbers of rears and line crossings did not decreasesignificantly showing that the drug was not sedative at this level.

As shown in FIG. 7, N,N'-di-(2-methylphenyl)guanidine did not affectsignificantly the number of rears, crossings, or the % time in the blackcompartment at the concentrations tested. This result is surprising inlight of the high sigma receptor binding of this compound (IC₅₀=32.0±1). Although the inventors do not wish to be bound by anyparticular theory, it would appear that DTG does not exhibit anxiolyticactivity in the in vivo assay since it is quickly metabolized andthereby deactivated by the animal.

Next, N-(adamantan-1-yl)-N'-(2-methylphenyl)guanidine,N,N'-di-(adamantan-1-yl)guanidine andN-(adamantan-1-yl)-N'-(o-iodophenyl)guanidine were orally administeredto the mice at a dose of 1 mg/kg. As shown in FIG. 4, orallyadministered N-(adamantan-1-yl)-N'-(2-methylphenyl)guanidine increasedsignificantly the proportion of time the mice spent in the larger,lighted area of the box. Moreover, the numbers of line crossings andrears in the light compartment increased at the expense of those in thedark compartment.

As shown in FIG. 8, N,N'-di-(adamantan-1-yl)guanidine andN-(adamantan-1-yl)-N'-(o-iodophenyl)guanidine caused an increase in thenumbers of line crossings and rears in the light compartment at theexpense of those in the dark compartment. Compared with controls, thenumbers of rears and line crossings did not decrease significantlyshowing that the two drugs were not sedative at the dosage leveladministered. The two drugs also caused an increase in the latency toentering the dark compartment in comparison to controls. Theseexperiments confirm thatN-(adamantan-1-yl)-N'-(2-methylphenyl)guanidine,N,N'-di-(adamantan-1-yl)guanidine andN-(adamantan-1-yl)-N'-(o-iodophenyl)guanidine have anxiolytic activitywhen orally administered.

As shown in FIG. 9, N-cyclohexyl-N'-(2-methylphenyl)guanidine increasessignificantly the proportion of time the mice spent in the larger,lighted area of the box. Moreover, the numbers of line crossings andrears in the light compartment increased in a dose-dependent manner atthe expense of those in the dark compartment. Compared with controls,the total number of rears and line crossings did not decreasesignificantly, showing that the drug was not sedative at the dosagelevels administered. The drug also caused an increase in the latency toentering the dark compartment compared to controls.

As shown in FIG. 10, N-((±)-endo-2-norbornyl)-N'-(2-iodophenyl)guanidinecaused a significant dose-dependent increase in the number of rears inthe light compartment at the expense of those in the dark compartment.Compared with controls, the total number of rears and line crossings didnot decrease significantly, showing that the drug was not sedative atthe dosage levels administered. The drug also caused a dose-dependentincrease in the latency to entering the dark compartment compared tocontrols.

As shown in FIG. 11, N-(exo-2-norbornyl)-N'-(2-methylphenyl)guanidinecaused an increase in the number of rears in the light compartment atthe expense of those in the dark compartment. Compared with controls,the total number of rears and line crossings did not decreasesignificantly, showing that the drug was not sedative at the dosagelevel administered. The drug also caused an increase in the latency toentering the dark compartment when administered at 0.1 mg/kg incomparison to controls.

As shown in FIG. 12, N-(adamantan-1-yl)-N'-cyclohexylguanidine caused anincrease in the number of rears in the light compartment at the expenseof those in the dark compartment. Compared with controls, the totalnumber of rears and line crossings did not change significantly, showingthat the drug was not sedative at the dosage levels administered. Thedrug also caused a dose-dependent increase in the latency to enteringthe dark compartment in comparison to controls.

As shown in FIG. 13, N-(cyclohexyl)-N'-(2-methylphenyl) guanidine, whenadministered orally, caused an increase in the total numbers of rears inthe light compartment at the expense of those in the dark compartment.Compared with controls, the number of rears and line crossings did notchange significantly, showing that the compound was not sedative at thedosage level administered. The compound also caused a slight increase inthe latency to entering the dark compartment in comparison to controls.However, a significant decrease in the percentage of time in the blackcompartment was observed.

As shown in FIG. 14, N-((±)-endo-2-norbornyl)-N'-2-iodophenyl)guanidine,administered orally, caused an increase in the number of rears in thelight compartment at the expense of those in the dark compartment.Compared with controls, the total number of rears and line crossings didnot decrease significantly, showing that the drug was not sedative atthe dosage level administered. The drug also caused an increase in thelatency to entering the dark compartment in comparison to controls and adecrease in the percentage time spent in the black compartment.

As shown in FIG. 15, N-(exo-2-norbornyl)-N'-(2-methylphenyl)guanidine,when administered orally, caused an increase in the number of rears inthe light compartment at the expense of those in the dark compartment.Compared with controls, the total number of rears and line crossings didnot decrease significantly, showing that the drug was not sedative atthe dosage levels administered. The drug also caused an increase in thelatency to entering the dark compartment in comparison with controls,and a decrease in the percentage time spent in the black compartment.

As shown in FIG. 16, N-(2-styrylphenyl)-N'-(2-iodophenyl)guanidine, anon-active control compound, did not affect the numbers of linecrossings or rears in the light compartment. Moreover, the drug actuallydecreased the latency to entering the dark compartment in comparisonwith controls. No significant change in the percentage time in the blackcompartment was observed.

Example 27 The Anxiolytic Potential of Diazepam andN-(adamantan-1-yl)-N'-(2-methylphenyl)guanidine in a Rat SocialInteraction Test

Male Hooded Lister rats (Glaxo bred, 200-250 g), were housed 5 to a cageand were kept in the laboratory environment for at least a week beforetesting. Rats paired in the test were taken from separate cages.

The compounds were screened for anxiolytic activity by Brenda Costallaccording to the disclosure of Jones, B. J. et al., Br. J. Pharmacol.93:985-993 (1988) and File, S. E. et al., Br. J. Pharmacol. 62:19-24(1978). The test arena consisted of an open-topped box, 62×62×33 cm witha 7×7 matrix of infra-red photocell beams in the walls, 2.5 cm from thefloor. Diazepam and N-(adamantan-1-yl)-N'-(2-methylphenyl)-guanidinewere tested by treating both members of a pair of rats with the sametreatment 40 min. before testing (Rats were placed singly in small cagesimmediately after dosing until they were tested).

Testing involved placing each member of a pair of rats in oppositecomers of the arena and then leaving them undisturbed for 10 min. whilerecording their behavior remotely on videotape. The behavioralassessments were made subsequently from the recordings. The time spentin social interaction was measured and expressed as a cumulative totalfor the 10 min session. The behaviors that comprised social interactionwere: following with contact, sniffing (but not sniffing of thehindquarters), crawling over and under, tumbling, boxing and grooming.

Intraperitoneally administered diazepam was tested over the dose rangeof 0.125-1 mg/kg. N-(adamantan-1-yl)-N'-(2-methylphenyl)guanidine wastested over the range of 0.001-0.1 mg/kg. The results appear in FIG. 17(n=6, P<0.001).

As shown in FIG. 17, both diazepam andN-(adamantan-1-yl)-N'-(2-methylphenyl)guanidine significantly increasedsocial interactions. However,N-(adamantan-1-yl)-N'-(2-methylphenyl)guanidine produced about the sameresult at one-tenth the dose of diazepam (1 mg/kg for diazepam and 0.1mg/kg for N-(adamantan-1-yl)-N'-(2-methylphenyl)guanidine). Theseresults provide strong indication thatN-(adamantan-1-yl)-N'-(2-methylphenyl)guanidine will have anxiolyticactivity in man, especially since the social interaction test in the ratis one of the most extensively validated tests (See File et al., supra).

The preceding examples can be repeated with similar success bysubstituting the generically or specifically described reactants and/oroperating conditions of this invention for those used in the precedingexamples.

From the foregoing description, one skilled in the art can easilyascertain the essential characteristics of this invention, and withoutdeparting from the spirit and scope thereof, can make various changesand modifications of the invention to adapt it to various usages andconditions.

What is claimed is:
 1. A pharmaceutical composition, in unit dosageform, which comprises, per unit dose, an amount effective to alter thesigma brain receptor modulated activity of a human being, of a watersoluble N,N'-disubstituted guanidine which displaces in vitroN,N'-di-(4-[³ H]-2-methylphenyl)guanidine bound to isolated mammalianbrain membrane, wherein said N,N'-disubstituted guanidine is selectedfrom the group consistingof:N-(adamantan-1-yl)-N'-(2-trifluoromethylphenyl)guanidine; andN-(adamantan-1-yl)-N'-(2-trifluoromethyl-4-fluorophenyl)guanidine. 2.The pharmaceutical composition of claim 1, in a form suitable for oraladministration.
 3. The pharmaceutical composition of claim 1, in a formsuitable for parenteral injection.
 4. The pharmaceutical composition ofclaim 1, wherein the effective amount of the disubstituted guanidineranges from about 0.1 mg to about 1 g per unit dose.
 5. A method oftreating a human being suffering from a psychotic mental illnessassociated with hallucinations, which comprising administering thereto,in an amount effective to ameliorate the hallucinations, anN,N'-disubstituted guanidine compound exhibiting high binding affinityto the sigma receptor and which is an antagonist to the sigma receptorbinding affinity of a hallucinogenic benzomorphan, wherein saidN,N'-disubstituted guanidine is selected from the group consistingof:N-(adamant-1-yl)-N'-(p-iodophenyl)guanidine;N-(adamantan-1-yl)-N'-(2-trifluoromethylphenyl)guanidine;N-(adamantan-1-yl)-N'-(2,4-difluorophenyl)guanidine; andN-(adamantan-1-yl)-N'-(2-trifluoromethyl-4-fluorophenyl)guanidine.
 6. Apharmaceutical composition comprising a pharmaceutically acceptablecarrier and an effective amount of a compound selected from the groupconsisting of:N-(adamantan-1-yl)-N'-(2-trifluoromethylphenyl)guanidine;and N-(adamantan-1-yl)-N'-(2-trifluoromethyl-4-fluorophenyl)guanidine.7. The pharmaceutical composition of claim 6 in a form suitable for oraladministration.
 8. The pharmaceutical composition of claim 6 in a formsuitable for parenteral administration.
 9. The pharmaceuticalcomposition of claim 6 wherein the effective amount of the compound isfrom about 0.1 mg to about 1 g per unit dose.
 10. A pharmaceuticalcomposition comprising a pharmaceutical carrier and an effective amountof N-(adamantan-1-yl)-N'-(2-trifluoromethyl-4-fluorophenyl)guanidine.11. The pharmaceutical composition of claim 10 in a form suitable fororal administration.
 12. The pharmaceutical composition of claim 10 in aform suitable for parenteral administration.
 13. The pharmaceuticalcomposition of claim 10 wherein the effective amount of the compound isfrom about 0.1 mg to about 1 g per unit dose.
 14. A method of treating ahuman being suffering from a psychotic mental illness comprisingadministering to said human an effective amount of a compound selectedfrom the group consistingof:N-(adamant-1-yl)-N'-(p-iodophenyl)guanidine;N-(adamantan-1-yl)-N'-(2-trifluoromethylphenyl)guanidine;N-(adamantan-1-yl)-N'-(2,4-difluorophenyl)guanidine; andN-(adamantan-1-yl)-N'-(2-trifluoromethyl-4-fluorophenyl)guanidine. 15.The method of claim 14 where the compound is administered orally. 16.The method of claim 14 where the compound is administered parenterally.17. The method of claim 14 wherein an effective amount ofN-(adamantan-1-yl)-N'-(2-trifluoromethyl-4-fluorophenyl)guanidine isadministered to the human.
 18. The method of claim 17 where theN-(adamantan-1-yl)-N'-(2-trifluoromethyl-4-fluorophenyl)guanidine isadministered orally.
 19. The method of claim 17 where theN-(adamantan-1-yl)-N'-(2-trifluoromethyl-4-fluorophenyl)guanidine isadministered parenterally.